Screening Method for the Identification of Agents Capable of Activating CD4+CD25+ Regulatory T-Cells Through Interactions with the HIV-1 GP120 Binding Site on CD4

ABSTRACT

The present invention relates specific activation of a regulatory T cell via a specific CD4 epitope and uses thereof, e.g. for the treatment of an autoimmune disease or an allergy or asthma or graft rejection or tolerance induction.

PRIORITY CLAIM

In accordance with 37 C.F.R. 1.76, a claim of priority is included in anApplication Data Sheet filed concurrently herewith. Accordingly, thepresent invention claims priority as a divisional of U.S. National Phasepatent application Ser. No. 12/525,142; entitled “SCREENING METHOD FORTHE IDENTIFICATION OF AGENTS CAPABLE OF ACTIVATING CD4⁺CD25⁺REGULATORYT-CELLS THROUGH INTERACTIONS WITH THE HIV-1 GP120 BINDING SITE ON CD4”,filed Oct. 7, 2009; which claims priority to International PCT PatentApplication No. PCT/EP2008/051144, entitled “SPECIFIC ACTIVATION OF AREGULATORY T CELL AND ITS USE FOR TREATMENT OF ASTHMA, ALLERGIC DISEASE,AUTOIMMUNE DISEASE, GRAFT REJECTION AND FOR TOLERANCE INDUCTION”, filedJan. 30, 2008, which claims priority to European Patent Application No.07101604.2, entitled “SPECIFIC ACTIVATION OF A REGULATORY T CELL AND ITSUSE FOR TREATMENT OF ASTHMA, ALLERGIC DISEASE, AUTOIMMUNE DISEASE, GRAFTREJECTION AND FOR TOLERANCE INDUCTION”, filed Feb. 1, 2007 and07122424.0, entitled “SPECIFIC ACTIVATION OF A REGULATORY T CELL AND ITSUSE FOR TREATMENT OF ASTHMA, ALLERGIC DISEASE, AUTOIMMUNE DISEASE, GRAFTREJECTION AND FOR TOLERANCE INDUCTION”, filed Dec. 5, 2007. The contentsof the above referenced applications are incorporated herein byreference.

INTRODUCTION

Asthma, an allergic disease, transplant rejection and an autoimmunedisease have one fundamental principle in common, they all are triggeredby an imbalanced immune system which reacts hyperactive against aspecific exogenic and/or endogenic challenge and therewith contributessignificantly to the disease status.

It is generally accepted that such aberrations of the immune system havea common pathophysiological mechanism triggered by hyper-responsiveeffector T cells playing a central role in immune reactivity. Effector Tcell-directed immunomodulation therefore is the key to successfultreatment of asthma, an autoimmune condition, prevention of graft vs.host disease (GVHD) and prevention of graft rejection.

T lymphocytes, designated as regulatory T cells (“Treg cells”) controlimmune responses by suppressing the effector function of CD4+ T cellsand CD8⁺ T cells (Shevach 2002). Different subsets of Treg cells havebeen described. These include but are not limited to (i) CD4⁺CD25⁺Tregcells—also designated as “naturally occurring Treg cells” (Sakaguchi2005), (ii) Tr1 (Roncarolo et al. 2001) and (iii) Th3 (Weiner 2001). Tr1and Th3 are induced in the periphery, whereas CD4⁺CD25⁺ Treg cellsdevelop in the thymus and constitute 5-10% of peripheral CD4⁺ T cells inhealthy man. At least in vitro these cells are anergic, produce minimalamounts of cytokines and exert their suppressive effects only uponstimulation and in a strictly cell-contact dependent manner. Tr1 and Th3exert their suppressive activity by production of IL-10 and TGF-beta,respectively (Shevach 2002).

Genetic defects that primarily affect Treg cell development or functionshould cause autoimmune and inflammatory aberrations. IPEX syndrome(immunodysregulation, polyendo-crinopathy and enteropathy, X-linked), arare recessive disorder in humans is caused by a mutation in the gene ofthe transcription factor FOXP3 and subsequent absence of Treg cells.IPEX shows aggressive autoimmunity, severe eczema, elevated IgE levels,eosinophilia and food allergies and early death (Fontenot and Rudensky2005).

Data from the literature show that Treg cells play an important role inasthma and autoimmune diseases and have a potential for treatment ofGVHD and therewith transplantation tolerance (Robinson 2004, Sakaguchi2005).

Therefore, attempts have been started to use Treg cells as a therapeuticagent for patients with established autoimmune disease (Horwitz et al.2003). It is believed that said patients lack sufficient Treg cells ordo have impaired Treg cell function resulting in misdirected anduncontrolled effector T cell activity. The previous thinking for solvingthis problem is administering Treg cells to a said patient. Since Tregcells are rare in peripheral blood clinical application of human Tregcells depends on highly expensive ex vivo expansion of Treg cells(Hoffmann et al. 2004, Horwitz et al. 2003, Tang et al. 2003, Zheng etal. 2004). Bluestone and Tang went one step further: They are trying tosolve the problem by not only increasing the amount of Treg cells fortherapy but enhancing suppressive activity of Treg cells by activatingTreg cells via triggering of the T cell receptor (TCR) by an anti-CD3antibody (Bluestone and Tang 2004). This approach resembles by far noTreg cell specific activation as anti-CD3 activates all T cellreceptor-expressing cells which bears the obstacle that anti-CD3treatment induces effector T cell function probably leading touncontrolled proliferation and non-specific pro-inflammatory cytokineproduction and exaggeration of pathology. To circumvent this unwantedtriggering of effector T cells Treg cells have to be highly purified andactivated ex vivo with anti-CD3 which again is a highly expensive andlaborious procedure. In addition, the absence of a Treg cell specificmarker makes it difficult to achieve high purity of Treg cells.

Therefore, it is a primary goal to identify a substance which canactivate Treg cells specifically without stimulating the immune systemany further and therefore would provide a basis for a direct in vivoapplication without money consuming ex vivo treatments.

There is some information in the prior art how Treg cells possibly canbe activated, e.g. nonspecifically via CD3 (Thornton and Shevach 1998),or via CD28/B7 pathway (Shevach 2002, Bluestone and Tang 2004, Hunig andDennehy 2005), or via CD4 (WO04083247). So far all this solutions do notresult in a specific Treg cell activation.

DESCRIPTION OF THE INVENTION

The present invention bases on the new finding that a specific epitopeof CD4 triggers activation of Treg cells. Said epitope overlaps with theknown HIV-1 gp120-binding site but surprisingly binding to this sitecauses Treg cell activation. This finding was totally unexpected for thefollowing reasons: Until now Treg cell activation via CD4 was attributedto a different epitope to which the monoclonal antibody BF5 (WO04083247)binds.—Carriere et al., 1995 have investigated that the binding site ofBF5 on CD4 is completely independent of the HIV-1 gp120-bindingsite.—Unexpectedly, despite reports in the literature on directanergizing and blocking of CD4 on T cell function by HIV-1 gp120(Diamond et al. 1988), we found an activating property of HIV-1 gp120 onCD4 of Treg cells.

The present invention discloses a physiologically active CD4 epitope ona Treg cell which triggers suppressive activity of a Treg cell. Theepitope of the present invention has been identified as a region on thehuman CD4 protein (SEQ ID NO.: 2) spanning amino acid position No. 54 to84 of SEQ ID NO.:2. Those 31 amino acids are explicitly given in SEQ IDNO.:1.

A preferred peptide which is a CD4 fragment according to the presentinvention is selected from a group consisting of an isolated peptidespanning amino acid No. 1-31 of SEQ ID NO 1, No. 26 to 458 of SEQ IDNO.: 2, No. 26 to 419 of SEQ ID NO.:2, No. 26 to 207 of SEQ ID NO.: 2,No. 26 to 131 of SEQ ID NO.: 2 and No. 46 to 89 of SEQ ID NO.: 2. Allsaid peptides are additionally characterized in that they all do harborthe critical amino acid Phenylalanine at residue 68 of SEQ ID NO.: 2.

The finding that the epitope given in SEQ ID NO.:1 is a key to activatea Treg cell provides a basis for several uses, e.g.:

Methods for identification of a substance which can activate aregulatory T cell (Treg cell). Such a substance is designated “Treg cellactivator” of the present invention which is useful for the treatment ofa disease of the invention which is a disease in which increase ofactivated regulatory T cells (Treg cells) can improve clinical picturewherein such a disease is (i) a non-autoimmune inflammatory disease:asthma, allergic asthma, respiratory allergy, allergicrhinoconjunctivitis, allergic alveolitis, contact allergy, atopicdermatitis, neurodermatitis, food allergy, graft-versus-host disease,non-autoimmune inflammatory bowel disease, acute respiratory distresssyndrome, acute inflammatory pancreatitis, burns, wound healing, skinscarring disorders, sarcoidosis, Behcet's disease or Sweet's syndrome;(ii) an autoimmune inflammatory disease: rheumatoid arthritis, rheumaticfever, systemic lupus erythematosus, ulcerative colitis, Crohn'sdisease, autoimmune inflammatory bowel disease, diabetes type I,gastritis, autoimmune atrophic gastritis, autoimmune hepatitis,Hashimoto's thyroiditis, thyreoiditis, multiple sclerosis, myastheniagravis, autoimmune haemolytic anemia, Addison's disease, scleroderma,Goodpasture's syndrome, Guillain-Barre syndrome, Graves' disease,glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid, vitiligo,idiopathic leukopenia, Sjogren's syndrome or Wegener's granulomatosis;(iii) an inflammatory disease due to organ transplantation; (iv) a bonemarrow transplantation; or (v) a disease due to exogenously administeredself or exogenously administered non-autologous recombinant polypeptide.

A “substance” of the present invention can be used in a method accordingto the present invention. The meaning of the term substance according tothe present invention includes but is not limited to a peptide, ascaffolded peptide, an antibody, a fragment of an antibody, a nucleicacid molecule, a ribozyme, an organic compound or an inorganic compound.

In a second aspect the new epitope serves as the basis for the synthesisof a new tool which can be used e.g. in a competition assay or screeningassay according to the present invention for determining whether asubstance can activate a regulatory T cell (Treg cell) via interactionwith the epitope as set forth in SEQ ID NO.:1. Such a tool is a peptideof the present invention which is an isolated peptide spanning aminoacid No. 1-31 of SEQ ID NO 1, or No. 26 to 458 of SEQ ID NO.: 2, or No.26 to 419 of SEQ ID NO.:2, or No. 26 to 207 of SEQ ID NO.: 2, or No. 26to 131 of SEQ ID NO.: 2 or No. 46 to 89 of SEQ ID NO.: 2, or is anisolated peptide spanning amino acid No. 1 to 31 of SEQ ID NO.: 1 andhaving additional up-stream and/or downstream amino acids with theprerequisite that the additional amino acids do not hinder binding of asubstance to the amino acid stretch as set forth in SEQ ID NO.: 1. Apreferred peptide according to the present invention consist of thepeptide as set forth in SEQ ID NO.:1 and additionally consist of anadditional up-stream amino acid or amino acid stretch which is selectedfrom a group consisting of the amino acid or amino acid stretch as setforth in SEQ ID NO.: 2 at position 53, at position 52-53, at position51-53, at position 50-53, at position 49-53, at position 48-53, atposition 47-53, at position 46-53, at position 45-53, at position 44-53,at position 43-53, at position 42-53, at position 41-53, at position40-53, at position 39-53, at position 38-53, at position 37-53, atposition 36-53, at position 35-53, at position 34-53, at position 33-53,at position 32-53, at position 31-53, at position 30-53, at position29-53, at position 28-53, at position 27-53, at position 26-53, atposition 25-53, at position 24-53, at position 23-53, at position 22-53,at position 21-53, at position 20-53, at position 19-53, at position18-53, at position 17-53, at position 16-53, at position 15-53, atposition 14-53, at position 13-53, at position 12-53, at position 11-53,at position 10-53, at position 9-53, at position 8-53, at position 7-53,at position 6-53, at position 5-53, at position 4-53, at position 3-53,at position 2-53, and at position 1-53.

A further preferred peptide according to the present invention consistsof the peptide which is mentioned as “preferred peptide” in theparagraph above and additionally consists of at least one additionaldownstream amino acid as given in SEQ ID NO.: 2 at position 85, oradditionally consists of amino acids as given in SEQ ID NO.:2 atposition 85 to n, wherein n is an integer between 86-458, i.e. position85 to 86, 85 to 87, 85 to 88, 85 to 89, 85 to 90, 85 to 91, 85 to 92, 85to 93, 85 to 94, 85 to 95, 85 to 96, 85 to 97, 85 to 98, 85 to 99, 85 to100, 85 to 101, 85 to 102, 85 to 103, 85 to 104, 85 to 105, 85 to 106,85 to 107, 85 to 108, 85 to 109, 85 to 110, 85 to 111, 85 to 112, 85 to113, 85 to 114, 85 to 115, 85 to 116, 85 to 117, 85 to 118, 85 to 119,85 to 120, 85 to 121, 85 to 122, 85 to 123, 85 to 124, 85 to 125, 85 to126, 85 to 127, 85 to 128, 85 to 129, 85 to 130, 85 to 131, 85 to 132,85 to 133, 85 to 134, 85 to 135, 85 to 136, 85 to 137, 85 to 138, 85 to139, 85 to 140, 85 to 141, 85 to 142, 85 to 143, 85 to 144, 85 to 145,85 to 146, 85 to 147, 85 to 148, 85 to 149, 85 to 150, 85 to 151, 85 to152, 85 to 153, 85 to 154, 85 to 155, 85 to 156, 85 to 157, 85 to 158,85 to 159, 85 to 160, 85 to 161, 85 to 162, 85 to 163, 85 to 164, 85 to165, 85 to 166, 85 to 167, 85 to 168, 85 to 169, 85 to 170, 85 to 171,85 to 172, 85 to 173, 85 to 174, 85 to 175, 85 to 176, 85 to 177, 85 to178, 85 to 179, 85 to 180, 85 to 181, 85 to 182, 85 to 183, 85 to 184,85 to 185, 85 to 186, 85 to 187, 85 to 188, 85 to 189, 85 to 190, 85 to191, 85 to 192, 85 to 193, 85 to 194, 85 to 195, 85 to 196, 85 to 197,85 to 198, 85 to 199, 85 to 200, 85 to 201, 85 to 202, 85 to 203, 85 to204, 85 to 205, 85 to 206, 85 to 207, 85 to 208, 85 to 209, 85 to 210,85 to 211, 85 to 212, 85 to 213, 85 to 214, 85 to 215, 85 to 216, 85 to217, 85 to 218, 85 to 219, 85 to 220, 85 to 221, 85 to 222, 85 to 223,85 to 224, 85 to 225, 85 to 226, 85 to 227, 85 to 228, 85 to 229, 85 to230, 85 to 231, 85 to 232, 85 to 233, 85 to 234, 85 to 235, 85 to 236,85 to 237, 85 to 238, 85 to 239, 85 to 240, 85 to 241, 85 to 242, 85 to243, 85 to 244, 85 to 245, 85 to 246, 85 to 247, 85 to 248, 85 to 249,85 to 250, 85 to 251, 85 to 252, 85 to 253, 85 to 254, 85 to 255, 85 to256, 85 to 257, 85 to 258, 85 to 259, 85 to 260, 85 to 261, 85 to 262,85 to 263, 85 to 264, 85 to 265, 85 to 266, 85 to 267, 85 to 268, 85 to269, 85 to 270, 85 to 271, 85 to 272, 85 to 273, 85 to 274, 85 to 275,85 to 276, 85 to 277, 85 to 278, 85 to 279, 85 to 280, 85 to 281, 85 to282, 85 to 283, 85 to 284, 85 to 285, 85 to 286, 85 to 287, 85 to 288,85 to 289, 85 to 290, 85 to 291, 85 to 292, 85 to 293, 85 to 294, 85 to295, 85 to 296, 85 to 297, 85 to 298, 85 to 299, 85 to 300, 85 to 301,85 to 302, 85 to 303, 85 to 304, 85 to 305, 85 to 306, 85 to 307, 85 to308, 85 to 309, 85 to 310, 85 to 311, 85 to 312, 85 to 313, 85 to 314,85 to 315, 85 to 316, 85 to 317, 85 to 318, 85 to 319, 85 to 320, 85 to321, 85 to 322, 85 to 323, 85 to 324, 85 to 325, 85 to 326, 85 to 327,85 to 328, 85 to 329, 85 to 330, 85 to 331, 85 to 332, 85 to 333, 85 to334, 85 to 335, 85 to 336, 85 to 337, 85 to 338, 85 to 339, 85 to 340,85 to 341, 85 to 342, 85 to 343, 85 to 344, 85 to 345, 85 to 346, 85 to347, 85 to 348, 85 to 349, 85 to 350, 85 to 351, 85 to 352, 85 to 353,85 to 354, 85 to 355, 85 to 356, 85 to 357, 85 to 358, 85 to 359, 85 to360, 85 to 361, 85 to 362, 85 to 363, 85 to 364, 85 to 365, 85 to 366,85 to 367, 85 to 368, 85 to 369, 85 to 370, 85 to 371, 85 to 372, 85 to373, 85 to 374, 85 to 375, 85 to 376, 85 to 377, 85 to 378, 85 to 379,85 to 380, 85 to 381, 85 to 382, 85 to 383, 85 to 384, 85 to 385, 85 to386, 85 to 387, 85 to 388, 85 to 389, 85 to 390, 85 to 391, 85 to 392,85 to 393, 85 to 394, 85 to 395, 85 to 396, 85 to 397, 85 to 398, 85 to399, 85 to 400, 85 to 401, 85 to 402, 85 to 403, 85 to 404, 85 to 405,85 to 406, 85 to 407, 85 to 408, 85 to 409, 85 to 410, 85 to 411, 85 to412, 85 to 413, 85 to 414, 85 to 415, 85 to 416, 85 to 417, 85 to 418,85 to 419, 85 to 420, 85 to 421, 85 to 422, 85 to 423, 85 to 424, 85 to425, 85 to 426, 85 to 427, 85 to 428, 85 to 429, 85 to 430, 85 to 431,85 to 432, 85 to 433, 85 to 434, 85 to 435, 85 to 436, 85 to 437, 85 to438, 85 to 439, 85 to 440, 85 to 441, 85 to 442, 85 to 443, 85 to 444,85 to 445, 85 to 446, 85 to 447, 85 to 448, 85 to 449, 85 to 450, 85 to451, 85 to 452, 85 to 453, 85 to 454, 85 to 455, 85 to 456, 85 to 457,or 85 to 458.

Additionally, the finding that the epitope given in SEQ ID NO.:1 is akey to activate a Treg cell is a link between two different up to nowunrelated technical fields, namely that of (a) HIV-1 related diseaseswith (b) diseases according to the present invention e.g. autoimmunedisease, allergy, asthma, graft rejection and a diseases due to lackingimmunotolerance caused by organ transplantation or by therapeuticaladministration of a non-self or self biological entity to a human inneed thereof and therewith allows a bundle of new uses as explained inthe following:

The epitope given in SEQ ID NO.:1 is not only a further epitope whichcan be used to activate Treg cells. HIV-1 gp120 interacts with CD4 of Tcells and therewith enables virus entry into a CD4⁺ cell (Klatzmann etal. 1984). The finding that epitope given in SEQ ID NO.:1 harbors thehigh affinity binding site on CD4 to which human immune deficiency virus1 (HIV-1) glycoprotein gp120 binds (Jameson et al. 1988, Arthos et al.1989) offers a further advantage. It provides the basis to bringtogether the findings of two different unrelated technical fields thatof i.e. HIV-1 related diseases with diseases according to the presentinvention.

To alleviate the worldwide HIV-1 problem many efforts have been made toidentify a substance which is able to inhibit HIV-1 entry into a CD4⁺cell. As a result thereof, so called HIV-1 attachment or entryinhibitors are known in the art.

The keyhole which allows HIV-1 to enter the cell can be used as thekeyhole to activate Treg cells. Therefore, substances known in the artto interfere with HIV-1 attachment and cell entry (Markovic and Clouse2004, Castagna et al. 2005), like e.g. HIV-1 gp120 itself, derivativesthereof, peptidomimetics, antibodies, aptamers or any low molecularweight (LMW) compound directed against the binding site of HIV-1 gp120on CD4 could possibly be useful to activate a Treg cell and therewithcan be useful for the treatment of a disease according to the presentinvention (HIV-1 gp120 is well-known in the art and its amino acidsequence as well as the respective gene has been published since years(Muesing et al. 1985, Starcich et al. 1986, Jeffs et al. 1996).Additionally, methods for producing HIV-1 gp120 are known (Lasky et al.1986, Leonard et al. 1990, Culp et al. 1991, Jeffs et al. 1996).

A substance which can interfere with HIV-1 attachment and/or cell entryis commonly named HIV-1 attachment inhibitor or entry inhibitor. Such asubstance, do either bind to (i) HIV-1, or (ii) to CD4, or (iii) HIV-1and CD4 or (iv) co-receptor e.g. CCR5 or CXCR4. Such an inhibitor can beaccording to the present invention useful for the treatment of diseasesaccording to the present invention like e.g. an autoimmune disease, anallergy, asthma or GVHD if it exerts property (ii) or (iii) andactivates a Treg cell. To determine whether such an inhibitor can beuseful for a said disease the present invention discloses several assayswhich allow to determine whether a substance identified in the technicalfield of HIV-1 research to interfere with HIV-1 attachment and/or cellentry can be useful in the other above-mentioned technical fields likethat of autoimmune diseases or allergies or asthma or organtransplantation. Therefore, the present invention teaches a short cutfor identifying a substance which can be useful for the treatment of adisease according to the present invention.

Identification of a substance which can be used as a medicament in aspecific disease usually depends on resource consuming high through putscreenings (HTS). Determination whether a substance can activate a Tregcell currently depends on a cellular assay comprising a Treg cell. SinceTreg cells can only be provided in small amounts large screeningcampaigns or even an HTS therewith are not feasible today. The teachingof the present invention allows to circumvent this obstacles since thepresent invention allows to pre-select substances which possible canactivate a Treg cell. According to the present invention an appropriatepre-selected substance is (i) proven to interact at least with epitope(SEQ ID NO.:1), and/or is (ii) known from HIV-1 research as HIV-1attachment inhibitor or entry inhibitor or synthetic mimetics of the CD4binding site of HIV-1 gp120.

It is state of the art to determine whether a substance can interactwith a given peptide and therewith with a given epitope even in an HTSformat. Concerning the present invention this can be performed forexample in an in vitro competition type assay comprising a peptidespanning at least amino acids as set forth in SEQ ID NO.: 1 mixed withan unlabeled substance to be tested and subsequently with a labeledsubstance which is known to bind the peptide (e.g. HIV-1 gp120) underconditions which allow binding of the peptide with the labeledsubstance. A substance which interact with the peptide will compete withthe labeled substance and is identifiable by a rendered readout, whichcan be performed e.g. by measuring the bound or free labeled substance.

Such a type of assay for determining whether a substance can interactwith a specific peptide, i.e. epitope is not restricted to in vitroassays since cellular assays for achieving such an information on asubstance or other in vitro formats are well known in the art andbroadly used.

In one embodiment the present invention concerns a method fordetermining whether a substance which can interfere with the interactionof HIV-1 gp120 with CD4 can be useful for positively influence a diseasein which increase of activated regulatory T cells (Treg cells) canimprove clinical picture comprising:

-   -   (a) providing a solution comprising a Treg cell, wherein a Treg        cell is preferably a CD4⁴CD25⁺ Treg cell or a Tr1 cell or a Th3        cell. Said solution does more preferably comprise additionally        an inactivated syngenic CD3-depleted PBMC (peripheral blood        mononuclear cell which has preferably been inactivated via        irradiation or via mitomycin C) or a dendritic cell (DC) and an        allogeneic CD8⁺ T cell or an allogeneic CD4⁺ T cell,    -   (b) adding a substance to be tested under conditions which allow        interaction of the substance with a Treg cell,    -   (c) measuring whether a Treg cell has been activated, wherein an        activated Treg cell identifies the substance as a Treg cell        activator.    -   Said measuring can be performed using a suitable read-out system        such as:        -   (i) measuring whether a CD8⁺ T cell has been            suppressed—which preferably can be determined by measuring            inhibited proliferation of the CD8⁺ T cell or by measuring            reduced CD25 expression of the CD8⁺ T cell, or by measuring            inhibited cytokine production of the CD8⁺ T cell wherein a            suitable cytokine is IFNγ or IL2, or TNFα—wherein a            suppressed CD8⁺ T cell identifies an activated Treg cell and            therewith identifies the substance as a Treg cell activator,            or by (ii) measuring whether a CD4⁺ T cell has been            suppressed—which preferably can be determined by measuring            inhibited proliferation of the CD4⁺ T cell or by measuring            reduced CD25 expression of the CD4⁺ T cell, or by measuring            inhibited cytokine production of the CD4⁺ T cell wherein a            suitable cytokine is IFNγ or IL2, or TNFα—wherein a            suppressed CD4⁺ T cell identifies an activated Treg cell and            therewith identifies the substance as a Treg cell activator,            or by (iii) measuring the amount of intracellular cAMP (i.e.            cytosolic cAMP) and wherein an increased amount of            intracellular cAMP is indicative for an activated Treg cell            and therewith identifies the substance as a Treg cell            activator.

A disease according to the method above in which increase of activatedregulatory T cells (Treg cells) can improve clinical picture is (i) anon-autoimmune inflammatory disease: asthma, allergic asthma,respiratory allergy, allergic rhinoconjunctivitis, allergic alveolitis,contact allergy, atopic dermatitis, neurodermatitis, food allergy,graft-versus-host disease, non-autoimmune inflammatory bowel disease,acute respiratory distress syndrome, acute inflammatory pancreatitis,burns, wound healing, skin scarring disorders, sarcoidosis, Behcet'sdisease or Sweet's syndrome; (ii) an autoimmune inflammatory disease:rheumatoid arthritis, rheumatic fever, systemic lupus erythematosus,ulcerative colitis, Crohn's disease, autoimmune inflammatory boweldisease, diabetes type I, gastritis, autoimmune atrophic gastritis,autoimmune hepatitis, Hashimoto's thyroiditis, thyreoiditis, multiplesclerosis, myasthenia gravis, autoimmune haemolytic anemia, Addison'sdisease, scleroderma, Goodpasture's syndrome, Guillain-Barre syndrome,Graves' disease, glomerulonephritis, psoriasis, pemphigus vulgaris,pemphigoid, vitiligo, idiopathic leukopenia, Sjogren's syndrome orWegener's granulomatosis; (iii) an inflammatory disease due to organtransplantation; (iv) a bone marrow transplantation; or (v) a diseasedue to exogenously administered self or exogenously administerednon-autologous recombinant polypeptide.

As the readout systems as mentioned in (i) and (ii) above are methods inwhich several steps have to be performed a specific new test system hasbeen invented to determine in only one step whether a Treg cell has beenactivated which is mentioned in (iii) above. Basing on the surprisingfinding that the activation status of a Treg cell strongly correlateswith the amount of intracellular cAMP the present invention provides fora specific method for determining whether a Treg cell has been activatedwhich comprises:

-   -   (a) providing a first solution comprising a Treg cell,    -   (b) providing a second solution comprising a Treg cell,    -   (c) manipulation the first solution by adding at least a test        substance,    -   (d) determining the amount of intracellular cAMP of the first        and the second solution    -   wherein an increased amount of intracellular cAMP of the first        solution is indicative for an activated Treg cell.

In a more preferred method the solution of (a) does not only contain aTreg cell but also an inactivated syngenic CD3-depleted PBMC (peripheralblood mononuclear cell) or a dendritic cell (DC) and an allogeneic CD8⁺T cell or an allogeneic CD4⁺ T cell. These cells when combined in onesolution with a activated Treg cell do additionally increaseintracellular cAMP amount and therewith lead to a more sensitive readoutsystem.

In a further embodiment the present invention concerns a method fordetermining whether a substance can activate a Treg cell via interactionwith the HIV-1 gp120-binding site of CD4, comprising:

-   -   (a) pre-selecting a substance which can interact with the HIV-1        gp120-binding site of CD4 (for a method for pre-selection please        see below),    -   (b) providing a solution comprising a Treg cell wherein a Treg        cell is preferably a CD4⁺CD25⁺ Treg cell or a Tr1 cell or a Th3        cell. Said solution does more preferably comprise additionally        an inactivated syngenic CD3-depleted PBMC (peripheral blood        mononuclear cell) which has preferably been inactivated via        irradiation or via mitomycin C) or a dendritic cell (DC) and an        allogeneic CD8⁺ T cell or an allogeneic CD4⁺ T cell,    -   (c) adding a pre-selected substance according to (a) under        conditions which allow interaction of the substance with a Treg        cell,    -   (d) measuring whether a Treg cell has been activated, wherein an        activated Treg cell identifies the substance as a Treg cell        activator. Said measuring can be performed using a suitable        read-out system such as:        -   (i) measuring whether a CD8⁺ T cell has been            suppressed—which preferably can be determined by measuring            inhibited proliferation of the CD8⁺ T cell or by measuring            reduced CD25 expression of the CD8⁺ T cell, or by measuring            inhibited cytokine production of the CD8⁺ T cell wherein a            suitable cytokine is IFNγ or IL2, or TNFα—wherein a            suppressed CD8⁺ T cell identifies an activated Treg cell and            therewith identifies the substance as a Treg cell activator,            or by        -   (ii) measuring whether a CD4⁺ T cell has been            suppressed—which preferably can be determined by measuring            inhibited proliferation of the CD4⁺ T cell or by measuring            reduced CD25 expression of the CD4⁺ T cell, or by measuring            inhibited cytokine production of the CD4⁺ T cell wherein a            suitable cytokine is IFNγ or IL2, or TNFα—wherein a            suppressed CD4⁺ T cell identifies an activated Treg cell and            therewith identifies the substance as a Treg cell activator,            or by    -   (iii) measuring the amount of intracellular cAMP and wherein an        increased amount of intracellular cAMP is indicative for an        activated Treg cell and therewith identifies the substance as a        Treg cell activator.

The present invention also provides for a method which allow to identifya substance which can interact with the HIV-1 gp120-binding site of CD4and therewith can be used for pre-selecting a substance which caninteract with the HIV-1 gp120-binding site of CD4.

The method comprises:

-   -   (a) providing a first solution comprising CD4,    -   (b) providing a second solution comprising CD4,    -   (c) adding to the first solution a substance to be tested and        HIV-1 gp120 under conditions which allow binding of HIV-1 gp120        with CD4,    -   (d) adding to the second solution HIV-1 gp120 under conditions        like (c) allowing binding of HIV-1 gp120 with CD4,    -   (e) measuring in the first and in the second solution whether        the HIV-1 gp120 has bound to the CD4 wherein a reduced amount of        bound HIV-1 gp120 in the first solution indicates that the        substance can interact with HIV-1 gp120-binding site of CD4.

This pre-selecting method can additionally be performed according to thepresent invention if instead of CD4 a peptide is used which comprisesthe amino acid stretch as set forth in SEQ ID NO.1 or instead of CD4 apeptide is used as set forth in SEQ ID NO.:1. In a more preferred methodaccording to the present invention instead of HIV-1 gp120 a peptide isused selected from a group consisting of a peptide comprising the aminoacid sequence as set forth in SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.:5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, and SEQID NO.: 10 or a peptide is used selected from a group consisting of apeptide consisting of the amino acid sequence as set forth in SEQ IDNO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQID NO.: 8, SEQ ID NO.: 9, and SEQ ID NO.: 10.

In a further embodiment the present invention concerns a method fordetermining whether a substance can activate a regulatory T cell (Treg)via interaction with the epitope as set forth in SEQ ID NO: 1comprising:

-   -   (a) providing a first solution comprising a Treg cell, wherein a        Treg cell is preferably a CD4⁺CD25⁺ Treg cell or a Tr1 cell or a        Th3 cell. Said solution does more preferably comprise        additionally an inactivated syngenic CD3-depleted PBMC        (peripheral blood mononuclear cell which has preferably been        inactivated via irradiation or via mitomycin C) or a dendritic        cell (DC) and an allogeneic CD8⁺ T cell or an allogeneic CD4⁺ T        cell,    -   (b) adding a substance to be tested under conditions which allow        interaction of the substance with a Treg cell,    -   (c) measuring whether a Treg cell of the first solution has been        activated,    -   (d) providing a second solution comprising a Treg cell, wherein        a Treg cell is preferably a CD4⁺CD25⁺Treg cell or a Tr1 cell or        a Th3 cell. Said solution does more preferably comprise        additionally an inactivated syngenic CD3-depleted PBMC        (peripheral blood mononuclear cell which has preferably been        inactivated via irradiation or via mitomycin C) or a dendritic        cell (DC) and an allogeneic CD8⁺ T cell or an allogeneic CD4⁺ T        cell, (e) adding the substance to be tested and a peptide        comprising an amino acid sequence as set forth in SEQ ID NO: 1        under conditions like (b),    -   (f) measuring whether a Treg cell of the second solution has        been activated,    -   Said measuring can be performed using a suitable read-out system        such as:        -   (i) measuring whether a CD8⁺ T cell has been            suppressed—which preferably can be determined by measuring            inhibited proliferation of the CD8⁺ T cell or by measuring            reduced CD25 expression of the CD8⁺ T cell, or by measuring            inhibited cytokine production of the CD8⁺ T cell wherein a            suitable cytokine is IFNγ or IL2, or TNFα wherein a            suppressed CD8⁺ T cell identifies an activated Treg cell and            therewith identifies the substance as a Treg cell activator,            or by        -   (ii) measuring whether a CD4⁺ T cell has been            suppressed—which preferably can be determined by measuring            inhibited proliferation of the CD4⁺ T cell or by measuring            reduced CD25 expression of the CD4⁺ T cell, or by measuring            inhibited cytokine production of the CD4⁺ T cell wherein a            suitable cytokine is IFN-γ or IL2, or TNFα wherein a            suppressed CD4⁺ T cell identifies an activated Treg cell and            therewith identifies the substance as a Treg cell activator,            or by        -   (iii) measuring the amount of intracellular cAMP and wherein            an increased amount of intracellular cAMP is indicative for            an activated Treg cell and therewith identifies the            substance as a Treg cell activator;        -   (g) comparing results obtained from (c) with those obtained            from (0 wherein a reduced activation of (0 identifies the            substance as a Treg cell activator which activates a Treg            cell via interaction with the epitope as set forth in SEQ ID            NO: 1.

In a more preferred method in step (e) of above mentioned method thepeptide used is selected from a group consisting of an isolated peptidespanning amino acid No. 1-31 of SEQ ID NO 1, No. 26 to 458 of SEQ IDNO.: 2, No. 26 to 419 of SEQ ID NO.:2, No. 26 to 207 of SEQ ID NO.: 2,No. 26 to 131 of SEQ ID NO.: 2 and No. 46 to 89 of SEQ ID NO.: 2 or thepeptide used is an isolated peptide spanning amino acid No. 1 to 31 ofSEQ ID NO.: 1 and having additional up-stream and/or downstream aminoacids with the prerequisite that the additional amino acids do nothinder binding of a substance to the amino acid stretch as set forth inSEQ ID NO.:1. Said additional up-stream amino acid or amino acid stretchis preferably selected from a group consisting of the amino acid oramino acid stretch as set forth in SEQ ID NO.: 2 at position 53, atposition 52-53, at position 51-53, at position 50-53, at position 49-53,at position 48-53, at position 47-53, at position 46-53, at position45-53, at position 44-53, at position 43-53, at position 42-53, atposition 41-53, at position 40-53, at position 39-53, at position 38-53,at position 37-53, at position 36-53, at position 35-53, at position34-53, at position 33-53, at position 32-53, at position 31-53, atposition 30-53, at position 29-53, at position 28-53, at position 27-53,at position 26-53, at position 25-53, at position 24-53, at position23-53, at position 22-53, at position 21-53, at position 20-53, atposition 19-53, at position 18-53, at position 17-53, at position 16-53,at position 15-53, at position 14-53, at position 13-53, at position12-53, at position 11-53, at position 10-53, at position 9-53, atposition 8-53, at position 7-53, at position 6-53, at position 5-53, atposition 4-53, at position 3-53, at position 2-53, and at position 1-53.A more preferred peptide does additionally comprise downstream one ormore amino acids as set forth in SEQ ID NO.: 2 at position 85, or atposition 85 to n, wherein n is an integer between 86-458, i.e. position85 to 86, 85 to 87, 85 to 88, 85 to 89, 85 to 90, 85 to 91, 85 to 92, 85to 93, 85 to 94, 85 to 95, 85 to 96, 85 to 97, 85 to 98, 85 to 99, 85 to100, 85 to 101, 85 to 102, 85 to 103, 85 to 104, 85 to 105, 85 to 106,85 to 107, 85 to 108, 85 to 109, 85 to 110, 85 to 111, 85 to 112, 85 to113, 85 to 114, 85 to 115, 85 to 116, 85 to 117, 85 to 118, 85 to 119,85 to 120, 85 to 121, 85 to 122, 85 to 123, 85 to 124, 85 to 125, 85 to126, 85 to 127, 85 to 128, 85 to 129, 85 to 130, 85 to 131, 85 to 132,85 to 133, 85 to 134, 85 to 135, 85 to 136, 85 to 137, 85 to 138, 85 to139, 85 to 140, 85 to 141, 85 to 142, 85 to 143, 85 to 144, 85 to 145,85 to 146, 85 to 147, 85 to 148, 85 to 149, 85 to 150, 85 to 151, 85 to152, 85 to 153, 85 to 154, 85 to 155, 85 to 156, 85 to 157, 85 to 158,85 to 159, 85 to 160, 85 to 161, 85 to 162, 85 to 163, 85 to 164, 85 to165, 85 to 166, 85 to 167, 85 to 168, 85 to 169, 85 to 170, 85 to 171,85 to 172, 85 to 173, 85 to 174, 85 to 175, 85 to 176, 85 to 177, 85 to178, 85 to 179, 85 to 180, 85 to 181, 85 to 182, 85 to 183, 85 to 184,85 to 185, 85 to 186, 85 to 187, 85 to 188, 85 to 189, 85 to 190, 85 to191, 85 to 192, 85 to 193, 85 to 194, 85 to 195, 85 to 196, 85 to 197,85 to 198, 85 to 199, 85 to 200, 85 to 201, 85 to 202, 85 to 203, 85 to204, 85 to 205, 85 to 206, 85 to 207, 85 to 208, 85 to 209, 85 to 210,85 to 211, 85 to 212, 85 to 213, 85 to 214, 85 to 215, 85 to 216, 85 to217, 85 to 218, 85 to 219, 85 to 220, 85 to 221, 85 to 222, 85 to 223,85 to 224, 85 to 225, 85 to 226, 85 to 227, 85 to 228, 85 to 229, 85 to230, 85 to 231, 85 to 232, 85 to 233, 85 to 234, 85 to 235, 85 to 236,85 to 237, 85 to 238, 85 to 239, 85 to 240, 85 to 241, 85 to 242, 85 to243, 85 to 244, 85 to 245, 85 to 246, 85 to 247, 85 to 248, 85 to 249,85 to 250, 85 to 251, 85 to 252, 85 to 253, 85 to 254, 85 to 255, 85 to256, 85 to 257, 85 to 258, 85 to 259, 85 to 260, 85 to 261, 85 to 262,85 to 263, 85 to 264, 85 to 265, 85 to 266, 85 to 267, 85 to 268, 85 to269, 85 to 270, 85 to 271, 85 to 272, 85 to 273, 85 to 274, 85 to 275,85 to 276, 85 to 277, 85 to 278, 85 to 279, 85 to 280, 85 to 281, 85 to282, 85 to 283, 85 to 284, 85 to 285, 85 to 286, 85 to 287, 85 to 288,85 to 289, 85 to 290, 85 to 291, 85 to 292, 85 to 293, 85 to 294, 85 to295, 85 to 296, 85 to 297, 85 to 298, 85 to 299, 85 to 300, 85 to 301,85 to 302, 85 to 303, 85 to 304, 85 to 305, 85 to 306, 85 to 307, 85 to308, 85 to 309, 85 to 310, 85 to 311, 85 to 312, 85 to 313, 85 to 314,85 to 315, 85 to 316, 85 to 317, 85 to 318, 85 to 319, 85 to 320, 85 to321, 85 to 322, 85 to 323, 85 to 324, 85 to 325, 85 to 326, 85 to 327,85 to 328, 85 to 329, 85 to 330, 85 to 331, 85 to 332, 85 to 333, 85 to334, 85 to 335, 85 to 336, 85 to 337, 85 to 338, 85 to 339, 85 to 340,85 to 341, 85 to 342, 85 to 343, 85 to 344, 85 to 345, 85 to 346, 85 to347, 85 to 348, 85 to 349, 85 to 350, 85 to 351, 85 to 352, 85 to 353,85 to 354, 85 to 355, 85 to 356, 85 to 357, 85 to 358, 85 to 359, 85 to360, 85 to 361, 85 to 362, 85 to 363, 85 to 364, 85 to 365, 85 to 366,85 to 367, 85 to 368, 85 to 369, 85 to 370, 85 to 371, 85 to 372, 85 to373, 85 to 374, 85 to 375, 85 to 376, 85 to 377, 85 to 378, 85 to 379,85 to 380, 85 to 381, 85 to 382, 85 to 383, 85 to 384, 85 to 385, 85 to386, 85 to 387, 85 to 388, 85 to 389, 85 to 390, 85 to 391, 85 to 392,85 to 393, 85 to 394, 85 to 395, 85 to 396, 85 to 397, 85 to 398, 85 to399, 85 to 400, 85 to 401, 85 to 402, 85 to 403, 85 to 404, 85 to 405,85 to 406, 85 to 407, 85 to 408, 85 to 409, 85 to 410, 85 to 411, 85 to412, 85 to 413, 85 to 414, 85 to 415, 85 to 416, 85 to 417, 85 to 418,85 to 419, 85 to 420, 85 to 421, 85 to 422, 85 to 423, 85 to 424, 85 to425, 85 to 426, 85 to 427, 85 to 428, 85 to 429, 85 to 430, 85 to 431,85 to 432, 85 to 433, 85 to 434, 85 to 435, 85 to 436, 85 to 437, 85 to438, 85 to 439, 85 to 440, 85 to 441, 85 to 442, 85 to 443, 85 to 444,85 to 445, 85 to 446, 85 to 447, 85 to 448, 85 to 449, 85 to 450, 85 to451, 85 to 452, 85 to 453, 85 to 454, 85 to 455, 85 to 456, 85 to 457,or 85 to 458.

In the context of the present invention new and known substances havebeen examined in the assays according to the present invention. As aresult thereof substances could be identified which can act as a Tregcell activator of the present invention, i.e. such a substance is ableto activate a Treg cell via interaction with the Treg cell epitope asset forth in SEQ ID NO:1 which could be proven in vivo. The presentinvention therefore provides for Treg cell activators.

Disclosed are Treg cell activators according to the invention which arestructurally a peptide or a polypeptide, preferably an antibody of abinding fragment thereof or a scaffolded peptide.

The present invention further concerns a new antibody or a bindingfragment thereof capable of binding to the peptide as set forth in SEQID NO.:1 with the proviso that the antibody or the antibody fragment isnot OKT4A, OKT4D4, OKTcdr4a and not Leu3. Said disclaimed antibodies allrelate to a totally different technical filed, namely HIV-1 researchwhich is not related to the activation of Treg cells and diseaseaccording to the present invention. Even the present invention combinesfor the first time the technical field of (a) HIV-1 related diseaseswith (b) diseases according to the present invention e.g. autoimmunedisease, allergy, asthma, graft rejection and a diseases due to lackingimmunotolerance caused by organ transplantation or by therapeuticaladministration of a non-self or self biological entity to a human inneed thereof.

A further preferred Treg cell activator peptide is a peptide selectedfrom a group consisting of a peptide comprising the amino acid sequenceas set forth in SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.:6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, and SEQ ID NO.: 10, oris a peptide selected from a group consisting of a peptide consisting ofthe amino acid sequence as set forth in SEQ ID NO.: 3, SEQ ID NO.: 4,SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.:9, and SEQ ID NO.: 10.

A more preferred Treg cell activator polypeptide is selected from agroup consisting of: HIV-1 gp120, NSC 13778 which chemical structure isdisclosed in Yang et al. 2005 on page 6124 in FIG. 1, peptide 2 whichpresents three HIV-1 gp120 fragments bound together throughcomformationally flexible scaffolds which chemical structure isdisclosed in Franke et al. 2007 on page 4 at the bottom of the rightcolumn, monoclonal antibody OKT4A which binds to the HIV-1 gp120-bindingregion of CD4 as disclosed in Mizukami et al. 1988 on page 9273 rightcolumn line 19, monoclonal antibody OKT4D which binds to the HIV-1gp120-binding region of CD4 as disclosed in Mizukami et al. 1988 on page9273 right column line 19, monoclonal antibody OKTcdr4a which derivesfrom the murine OKT4a as disclosed in Moreland et al. 1998 on page 222right column line 1, monoclonal antibody Leu3 which binds to epitopeoverlapping the HIV-1 gp120-binding site of CD4 as disclosed in Lohmannet al. 1992 on page 3248 left column line 7, and monoclonal antibodyMAX.12H5 which binds to the CDR2-like region of CD4 as disclosed inRepke et al. 1992 on page 1809 abstract line 11 and on page 1812 leftcolumn line 37.

Each of the above mentioned Treg cell activators according to thepresent invention can be used as a medicament and for the preparation ofa medicament for the treatment of a disease selected from a groupconsisting of (i) a non-autoimmune inflammatory disease: asthma,allergic asthma, respiratory allergy, allergic rhinoconjunctivitis,allergic alveolitis, contact allergy, atopic dermatitis,neurodermatitis, food allergy, graft-versus-host disease, non-autoimmuneinflammatory bowel disease, acute respiratory distress syndrome, acuteinflammatory pancreatitis, burns, wound healing, skin scarringdisorders, sarcoidosis, Behcet's disease, Sweet's syndrome; (ii) anautoimmune inflammatory disease: rheumatoid arthritis, rheumatic fever,systemic lupus erythematosus, ulcerative colitis, Crohn's disease,autoimmune inflammatory bowel disease, diabetes type I, gastritis,autoimmune atrophic gastritis, autoimmune hepatitis, Hashimoto'sthyroiditis, thyreoiditis, multiple sclerosis, myasthenia gravis,autoimmune haemolytic anemia, Addison's disease, scleroderma,Goodpasture's syndrome, Guillain-Barre syndrome, Graves' disease,glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid, vitiligo,idiopathic leukopenia, Sjogren's syndrome, Wegener's granulomatosis;(iii) an inflammatory disease due to organ transplantation; (iv) a bonemarrow transplantation; and (v) a disease due to exogenouslyadministered self or exogenously administered non-autologous recombinantpolypeptide.

Another embodiment of the present invention relates to a pharmaceuticalcomposition comprising at least one Treg cell activator according to thepresent invention—preferably HIV-1 gp120—as an active ingredient and canbe formulated in conventional manner. Methods for making suchformulations can be found in manuals, e.g. “Remington PharmaceuticalScience”. Examples for ingredients that are useful for formulating atleast one substance according to the present invention are also found inWO99/18193, which is hereby incorporated by reference.

The composition may be manufactured in a manner that is itself known,e.g. by mean of conventional mixing, dissolving, granulating,dragee-making, levitating, powdering, emulsifying, encapsulating,entrapping of lyophilizing processes.

In a further aspect the invention teaches a method for treating adisease which is characterized in that its clinical picture can beinfluenced positively by an increase of activated Treg cells whichmethod comprises administering to a being preferably a human being inneed of such a treatment a suitable amount of a pharmaceuticalcomposition comprising at least one Treg cell activator according to thepresent invention, preferably HIV-1 gp120 or HIV-1 gp120 derivedfragments and peptides thereof. The present invention provides thereforefor a method for treating (i) a non autoimmune inflammatory disease:asthma, allergic asthma, respiratory allergy, allergicrhinoconjunctivitis, allergic alveolitis, contact allergy, atopicdermatitis, neurodermatitis, food allergy, graft-versus-host disease,non-autoimmune inflammatory bowel disease, acute respiratory distresssyndrome, acute inflammatory pancreatitis, burns, wound healing, skinscarring disorders, sarcoidosis, Behcet's disease or Sweet's syndrome;(ii) an autoimmune inflammatory disease: rheumatoid arthritis, rheumaticfever, systemic lupus erythematosus, ulcerative colitis, Crohn'sdisease, autoimmune inflammatory bowel disease, diabetes type I,gastritis, autoimmune atrophic gastritis, autoimmune hepatitis,Hashimoto's thyroiditis, thyreoiditis, multiple sclerosis, myastheniagravis, autoimmune haemolytic anemia, Addison's disease, scleroderma,Goodpasture's syndrome, Guillain-Barre syndrome, Graves' disease,glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid, vitiligo,idiopathic leukopenia, Sjogren's syndrome or Wegener's granulomatosis;(iii) an inflammatory disease due to organ transplantation; (iv) a bonemarrow transplantation; or (v) a disease due to exogenously administeredself or exogenously administered non-autologous recombinant polypeptidewhich method comprises administering to a being in need of such atreatment a suitable amount of a pharmaceutical composition comprisingat least one Treg cell activator.

The present invention additionally, provides for a use of a Treg cellactivator according to the present invention for reducing and/orpreventing an unwanted immune reaction due to a exogenously administeredself or exogenously administered non-autologous recombinant polypeptideand provides for a method for reducing or preventing an unwanted immunereaction comprising: (a) adding a sufficient amount of at least one Tregcell activator according to the present invention to a non-human animal,preferably a non-human primate.

In a further embodiment the present invention provides for a test systemfor determining whether a substance is a Treg cell activator accordingto the present invention comprising at least

a) a Treg cell and

b) a peptide spanning at least the epitope as set forth in SEQ ID NO.:1.

In a preferred test system of the present invention a peptide of b) isselected from a group consisting of an isolated peptide spanning aminoacid No. 26 to 458 of SEQ ID NO.: 2, amino acid No. 26 to 419 of SEQ IDNO.: 2, amino acid No. 26 to 207 of SEQ ID NO.: 2, amino acid No. 26 to131 of SEQ ID NO.: 2, and amino acid No. 46 to 89 of SEQ ID NO.: 2. orthe peptide used is an isolated peptide spanning amino acid No. 1 to 31of SEQ ID NO.: 1 and having additional up-stream and/or downstream aminoacids with the prerequisite that the additional amino acids do nothinder binding of a substance to the amino acid stretch as set forth inSEQ ID NO.: 1. Said additional up-stream amino acid or amino acidstretch is preferably selected from a group consisting of the amino acidor amino acid stretch as set forth in SEQ ID NO.: 2 at position 53, atposition 52-53, at position 51-53, at position 50-53, at position 49-53,at position 48-53, at position 47-53, at position 46-53, at position45-53, at position 44-53, at position 43-53, at position 42-53, atposition 41-53, at position 40-53, at position 39-53, at position 38-53,at position 37-53, at position 36-53, at position 35-53, at position34-53, at position 33-53, at position 32-53, at position 31-53, atposition 30-53, at position 29-53, at position 28-53, at position 27-53,at position 26-53, at position 25-53, at position 24-53, at position23-53, at position 22-53, at position 21-53, at position 20-53, atposition 19-53, at position 18-53, at position 17-53, at position 16-53,at position 15-53, at position 14-53, at position 13-53, at position12-53, at position 11-53, at position 10-53, at position 9-53, atposition 8-53, at position 7-53, at position 6-53, at position 5-53, atposition 4-53, at position 3-53, at position 2-53, and at position 1-53.A more preferred peptide does additionally comprise downstream one ormore amino acids as set forth in SEQ ID NO.: 2 at position 85, or atposition 85 to n, wherein n is an integer between 86-458, i.e. position85 to 86, 85 to 87, 85 to 88, 85 to 89, 85 to 90, 85 to 91, 85 to 92, 85to 93, 85 to 94, 85 to 95, 85 to 96, 85 to 97, 85 to 98, 85 to 99, 85 to100, 85 to 101, 85 to 102, 85 to 103, 85 to 104, 85 to 105, 85 to 106,85 to 107, 85 to 108, 85 to 109, 85 to 110, 85 to 111, 85 to 112, 85 to113, 85 to 114, 85 to 115, 85 to 116, 85 to 117, 85 to 118, 85 to 119,85 to 120, 85 to 121, 85 to 122, 85 to 123, 85 to 124, 85 to 125, 85 to126, 85 to 127, 85 to 128, 85 to 129, 85 to 130, 85 to 131, 85 to 132,85 to 133, 85 to 134, 85 to 135, 85 to 136, 85 to 137, 85 to 138, 85 to139, 85 to 140, 85 to 141, 85 to 142, 85 to 143, 85 to 144, 85 to 145,85 to 146, 85 to 147, 85 to 148, 85 to 149, 85 to 150, 85 to 151, 85 to152, 85 to 153, 85 to 154, 85 to 155, 85 to 156, 85 to 157, 85 to 158,85 to 159, 85 to 160, 85 to 161, 85 to 162, 85 to 163, 85 to 164, 85 to165, 85 to 166, 85 to 167, 85 to 168, 85 to 169, 85 to 170, 85 to 171,85 to 172, 85 to 173, 85 to 174, 85 to 175, 85 to 176, 85 to 177, 85 to178, 85 to 179, 85 to 180, 85 to 181, 85 to 182, 85 to 183, 85 to 184,85 to 185, 85 to 186, 85 to 187, 85 to 188, 85 to 189, 85 to 190, 85 to191, 85 to 192, 85 to 193, 85 to 194, 85 to 195, 85 to 196, 85 to 197,85 to 198, 85 to 199, 85 to 200, 85 to 201, 85 to 202, 85 to 203, 85 to204, 85 to 205, 85 to 206, 85 to 207, 85 to 208, 85 to 209, 85 to 210,85 to 211, 85 to 212, 85 to 213, 85 to 214, 85 to 215, 85 to 216, 85 to217, 85 to 218, 85 to 219, 85 to 220, 85 to 221, 85 to 222, 85 to 223,85 to 224, 85 to 225, 85 to 226, 85 to 227, 85 to 228, 85 to 229, 85 to230, 85 to 231, 85 to 232, 85 to 233, 85 to 234, 85 to 235, 85 to 236,85 to 237, 85 to 238, 85 to 239, 85 to 240, 85 to 241, 85 to 242, 85 to243, 85 to 244, 85 to 245, 85 to 246, 85 to 247, 85 to 248, 85 to 249,85 to 250, 85 to 251, 85 to 252, 85 to 253, 85 to 254, 85 to 255, 85 to256, 85 to 257, 85 to 258, 85 to 259, 85 to 260, 85 to 261, 85 to 262,85 to 263, 85 to 264, 85 to 265, 85 to 266, 85 to 267, 85 to 268, 85 to269, 85 to 270, 85 to 271, 85 to 272, 85 to 273, 85 to 274, 85 to 275,85 to 276, 85 to 277, 85 to 278, 85 to 279, 85 to 280, 85 to 281, 85 to282, 85 to 283, 85 to 284, 85 to 285, 85 to 286, 85 to 287, 85 to 288,85 to 289, 85 to 290, 85 to 291, 85 to 292, 85 to 293, 85 to 294, 85 to295, 85 to 296, 85 to 297, 85 to 298, 85 to 299, 85 to 300, 85 to 301,85 to 302, 85 to 303, 85 to 304, 85 to 305, 85 to 306, 85 to 307, 85 to308, 85 to 309, 85 to 310, 85 to 311, 85 to 312, 85 to 313, 85 to 314,85 to 315, 85 to 316, 85 to 317, 85 to 318, 85 to 319, 85 to 320, 85 to321, 85 to 322, 85 to 323, 85 to 324, 85 to 325, 85 to 326, 85 to 327,85 to 328, 85 to 329, 85 to 330, 85 to 331, 85 to 332, 85 to 333, 85 to334, 85 to 335, 85 to 336, 85 to 337, 85 to 338, 85 to 339, 85 to 340,85 to 341, 85 to 342, 85 to 343, 85 to 344, 85 to 345, 85 to 346, 85 to347, 85 to 348, 85 to 349, 85 to 350, 85 to 351, 85 to 352, 85 to 353,85 to 354, 85 to 355, 85 to 356, 85 to 357, 85 to 358, 85 to 359, 85 to360, 85 to 361, 85 to 362, 85 to 363, 85 to 364, 85 to 365, 85 to 366,85 to 367, 85 to 368, 85 to 369, 85 to 370, 85 to 371, 85 to 372, 85 to373, 85 to 374, 85 to 375, 85 to 376, 85 to 377, 85 to 378, 85 to 379,85 to 380, 85 to 381, 85 to 382, 85 to 383, 85 to 384, 85 to 385, 85 to386, 85 to 387, 85 to 388, 85 to 389, 85 to 390, 85 to 391, 85 to 392,85 to 393, 85 to 394, 85 to 395, 85 to 396, 85 to 397, 85 to 398, 85 to399, 85 to 400, 85 to 401, 85 to 402, 85 to 403, 85 to 404, 85 to 405,85 to 406, 85 to 407, 85 to 408, 85 to 409, 85 to 410, 85 to 411, 85 to412, 85 to 413, 85 to 414, 85 to 415, 85 to 416, 85 to 417, 85 to 418,85 to 419, 85 to 420, 85 to 421, 85 to 422, 85 to 423, 85 to 424, 85 to425, 85 to 426, 85 to 427, 85 to 428, 85 to 429, 85 to 430, 85 to 431,85 to 432, 85 to 433, 85 to 434, 85 to 435, 85 to 436, 85 to 437, 85 to438, 85 to 439, 85 to 440, 85 to 441, 85 to 442, 85 to 443, 85 to 444,85 to 445, 85 to 446, 85 to 447, 85 to 448, 85 to 449, 85 to 450, 85 to451, 85 to 452, 85 to 453, 85 to 454, 85 to 455, 85 to 456, 85 to 457,or 85 to 458.

The following examples are meant to illustrate the present invention,however, shall not be construed as limitation. However, the Examplesdescribe most preferred embodiments of the invention.

EXAMPLES

(1) CD4/HIV-1 gp120 competition assay for determining whether asubstance can bind at least to a peptide spanning epitope as set fourthin SEQ ID NO.: 1. 96 well assay plates (Nunc, Germany) are coatedovernight at 4° C. with CD4 (sCD4, Immunodiagnostics, USA) 100 ng perwell in PBS, pH 7.4. Coated plates are saturated with PBS/3% BSA bufferand washed three times. To determine binding of a test substance sampleis added for 1 hour in different concentrations. No test substance isadded to control wells. After washing three times HIV-1 gp120-peroxidaseconjugate (Immunodiagnostics, USA) is added to the plate for 1 h.Unbound HIV-1 gp120-peroxidase conjugate is removed by washing threetimes. After washing, 3,3,5,5-tetramethylbenzidine chromogen substrate(Pierce, USA) for peroxidase is added and the optical density is read at450 nm. A substance which interacts with the CD4 HIV-1 gp120-bindingsite will block the binding of labeled HIV-1 gp120 and is identifiableby a reduced signal compared to the controls.

(2) Assay for determining whether e.g. HIV-1 gp120 or a substance whichcan interfere with HIV-1 gp120-binding to a CD4⁺CD25⁺ Treg can activatea CD4⁺CD25⁺ Treg and therewith can be useful in the treatment anautoimmune disease (e.g. Inflammatory Bowel Disease, Multiple Sclerosis,Rheumatoid Arthritis, Psoriasis, Diabetes Type I, Lupus Erythematosus,Phemphigus vulgaris, Thyreoiditis), other diseases with autoimmuneaspects in their pathogenesis such as vitiligo, atopic dermatitis, anallergy (e.g. Allergic rhinitis,), asthma (e.g. allergic asthma), GVHD(graft-versus-host disease), graft rejection.

(2.1) Method for isolation of cells P (2.1.1) Isolation of PBMC

The isolation procedure starts with PBMC (peripheral blood mononuclearcells) isolated by standard density gradient centrifugation from normalbuffy coat preparations of healthy volunteers. Alternatively, PBMCisolated from whole peripheral blood or leukapheresis products can beused.

Blood from buffy coats is diluted 1:1 with PBS (phosphate bufferedsaline) containing 0.2% Liquemine (Sodium-Heparin) and 2 mM EDTA at roomtemperature. The diluted blood is thoroughly pipetted onto preparedFicoll layers (30 ml diluted blood per 15 ml Ficoll layer per 50 mltube) and centrifuged for 15 min. (minute) at 200×g (with brake on) atroom temperature. 8-10 ml of the upper fluid are carefully removed andthe tubes centrifuged at 450×g for 15 min. at room temperature (withbrake off). PBMC are collected from the interphase of each gradient,washed three times with 50 ml PBS/1 mM EDTA separately, than pooled andwash two more times. Finally, PBMC are re-diluted in X-VIVO-15 (Cambrex,Verviers, Belgium) in cell culture medium and counted.

(2.1.2) Isolation of CD4⁺ T helper cells

For determining suppressive activity of Treg cells highly purified cellpopulations are needed. Therefore, antibody-coated magnetic beads areused (Miltenyi, Germany and or Dynal, Norway). Magnetic beads in thiscontext are paramagnetic particles that are coupled to specificmonoclonal antibodies. They are used to magnetically label the targetcell population. The antibody-coated magnetic beads bind to the targetcells. This labeled cell fraction is retained by magnetic force and canbe recovered subsequently highly purified (positive selection). Beforepositive isolation of CD4⁺CD25⁻ T helper cells PBMC are washed two timeswith 50 ml washing buffer according to the manufacturer's instructions(Miltenyi Germany). For isolation of CD4⁺ T cells, CD4 microbeads(Miltenyi Germany, 2-4 μL microbeads/10⁷ PBMC) are used according to themanufacturer's instructions. The CD4⁺ fraction is isolated using a MACSseparator (Miltenyi) according to the instructions of the manufacturer.MACS separator retains magnetic bead-labeled cells by magnetic force.Contaminating CD4⁺CD25⁺ Treg cells are depleted in a second step byusing CD25 Dynabeads, (Dynal, Norway) according to the instructions ofthe manufacturer (details see 2.1.4) using (0.5 beads/cell). Thisdepletion procedure results in highly purified CD4⁺CD25⁻ T helper cells(negative selection).

To circumvent CD4 antibodies binding to CD4⁺ cells (to avoid CD4⁻dependent pre activation) in the isolation procedure of CD4⁺ T cells,alternatively untouched CD4⁺ T helper cells are generated by using thenegative isolation kit (Miltenyi Germany) according to themanufacturer's instructions. Before negative isolation of CD4⁺ T helpercells PBMC are washed two times with 50 ml washing buffer according tothe manufacturer's instructions (Miltenyi Germany). For isolation ofCD4⁺ T helper cells, PBMC are incubated with a cocktail of biotinylatedCD45RO, CD8, CD14, CD16, CD19, CD56, CD36, CD123, anti-TCRγ/δ, andCD235a antibodies. These cells are subsequently magnetically labeledwith Anti-Biotin Microbeads for depletion.

The CD4⁺ fraction is isolated using a MACS separator (Miltenyi)according to the instructions of the manufacturer. MACS separatorretains magnetic bead-labeled cells by magnetic force. ContaminatingCD4⁺CD25⁺ Treg cells are depleted in a second step by using CD25Dynabeads, (Dynal, Norway) according to the instructions of themanufacturer (details see 2.1.4) using 0.5 beads/cell.

(2.1.3) Isolation of CD8⁺ T effector cells

Before positive isolation of CD8⁺ T effector cells PBMC are washed twotimes with 50 ml washing buffer according to the manufacturer'sinstructions (Miltenyi Germany). For isolation of CD8⁺CD25⁻ T cells, CD8microbeads (Miltenyi Germany, 2-4 μL microbeads/10⁷ PBMC) are usedaccording to the manufacturer's instructions. The CD8⁺ fraction isisolated using a MACS separator (Miltenyi) according to the instructionsof the manufacturer. Contaminating CD8⁺CD25⁺ T cells are depleted in asecond step by using CD25 Dynabeads, (Dynal, Norway) according to theinstructions of the manufacturer (details see 2.1.4) using (0.5beads/cell). This depletion procedure results in highly purifiedCD8⁺CD25⁻ T effector cells (negative selection).

(2.1.4) Isolation of CD4⁺CD25⁺ Treg cells

For Isolation of CD4⁺CD25⁺ Treg cells positive and negative selection iscombined. PBMC are washed with washing buffer according to themanufacturer's instructions (Miltenyi Germany) and subsequentlyincubated with CD25 microbeads (2 μL microbeads/10⁷ PBMC) for 20 min. at4° C. in isolation buffer (1×10⁸/ml) according to the manufacturer'sinstructions. Afterwards, the cells are washed two times in PBS. TheCD25⁺ fraction is isolated using a MACS separator (Miltenyi) accordingto the instructions of the manufacturer. The positively selected CD25⁺fraction contains 65-80% CD4⁺ T cells and 20-35% contaminating CD19⁺ Bcells, CD8⁺ T cells, and few CD14⁺ monocytes. The contaminating cellsare depleted with Dynabeads (Dynal, Norway). The following amounts ofbeads are used: CD19 Dynabeads: 2 beads/cell, CD8 Dynabeads: 3beads/cell, CD14 Dynabeads: 1 bead/cell. Collected Dynabeads are washedtwo times in 15 ml tubes with depletion buffer using the magneticparticle concentrator (Dynal) according to the manufacturer'sinstructions. The CD25⁺ PBMC fraction (5×10⁷/ml) is added in depletionbuffer and incubated for 20 min. at 4° C. on a shaker (sample mixer,Dynal). Contaminating cells are depleted according to the manufacturer'sinstructions by the use of the magnetic particle concentrator. Forhigher purity of CD4⁺CD25⁺ Treg cells Dynabeads depletion is repeatedonce (>98% after two rounds of depletion).

(2.1.5) Generation of monocyte-derived dendritic cells

Dendritic cells (DC) are generated from buffy coats of healthyvolunteers. PBMC (2.1.6) are plated in 6-well tissue culture plates at adensity of 15×10⁶ cells/well in 3 ml X-VIVO-15 (Cambrex, Verviers,Belgium) plus 1.5% heat-inactivated autologous plasma containing 800U/ml GM-CSF (Leukomax; Novartis, Basel, Switzerland) and 1,000 U/ml IL-4(Strathmann Biotec, Hamburg, Germany). Cultures are fed every other day(days 2, 4 and 6) by removing 1 ml of the medium and adding back 1 mlfresh medium with cytokines. On day 7, non-adherent cells were harvestedand transferred to new 6 well plates and cultured further on in thepresence of 10 ng/ml IL-1β, 10 ng/ml TNF-α, 1,000 U/ml IL-6 (all fromStrathmann, Biotech, Germany) and 1 μg/ml PGE₂ (Pharmacia-Upjohn,Uppsala, Sweden). Mature CD83⁺ DC are harvested at day 9 of culture.

(2.1.6) Isolation of CD3-depleted PBMC

T cells are depleted from PBMC with CD3 Dynabeads (Dynal, Norway) byusing 0.5 beads/cell. Collected Dynabeads are washed two times in 15 mltubes with depletion buffer using the magnetic particle concentrator(Dynal) according to the manufacturer's instructions. PBMC (5×10⁷/ml)are added in depletion buffer and incubated for 20 min. at 4° C. on ashaker (sample mixer, Dynal). CD3⁺ cells are depleted according to themanufacturer's instructions by the use of the magnetic particleconcentrator resulting in a purity of >98% CD3⁻ PBMC.

(2.1.7) Isolation of human CD25-depleted PBMC

PBMC are isolated according to (2.1.1). CD25-expressing regulatory Tcells in the PBMC preparation are depleted with CD25 Dynabeads (Dynal,Norway) according to the instructions of the manufacturer (details see2.1.4) by using 0.5 beads/cell. Collected Dynabeads are washed two timesin 15 ml tubes with depletion buffer using the magnetic particleconcentrator (Dynal) according to the manufacturer's instructions. PBMC(5×10⁷/ml) are added in depletion buffer and incubated for 20 min. at 4°C. on a shaker (sample mixer, Dynal). CD25⁺ cells are depleted accordingto the manufacturer's instructions by the use of the magnetic particleconcentrator resulting in a purity of >99% CD25-negative PBMC.

(2.2) Method for testing suppressive activity of CD4⁺CD25⁺ Treg cells

(2.2.1) Coculture suppression assay A: Mixed leukocyte reaction (MLR)Cocultures of CD4⁺ T helper cells or CD8⁺ T effector cells withCD4⁺CD25⁺ Treg cells and allogeneic DC have to be performed to analyzethe suppressive activity of CD4⁺CD25⁺ Treg cells on CD4⁺ T helper cellsor CD8⁺ T effector cells. Therefore, 1×10⁵/well CD4⁺ T helper cells(2.1.2) or CD8⁺ T effector cells (2.1.3) are cocultured with differentnumbers of CD4⁺CD25⁺ Treg cells (2.1.4; ratio 1:1 to 1:4) and 1×10⁴/wellDC in 96 well flat bottom culture plates in X-VIVO 15 (Cambrex,Verviers, Belgium) in the presence or absence of a CD4-binding compounde.g. HIV-1 gp120 (0.1-10 μg/ml). Mature dendritic cells (DC) generatedas described (2.1.5) are from the same donor as CD4⁺CD25⁺ Treg cells(syngenic) but allogeneic to the CD4⁺ T helper cells or CD8⁺ T effectorcells are used for T cell stimulation. In this assay, only CD4⁺ T helpercells or CD8⁺ T effector cells are activated by the allogeneic DC (MLR)resulting in a strong proliferation of the T cell subset. Non-activatedCD4⁺CD25⁺ Treg cells did not suppress this proliferation in absence of aTreg cell activating compound. A functional activation of CD4⁺CD25⁺ Tregcells by a CD4⁻ binding compound resulted in a reduced proliferation ofCD4⁺T helper cells or CD8⁺ T effector cells.

Proliferation is determined after 4 days of culture by adding 37 kBq3H-Thymindine (³H-Tdr) for additional 16 h.

(2.2.2) Coculture Suppression Assay B: Stimulation of CD8⁺ T effectorcells with allogeneic PBMC and CD4⁺CD25⁺ Treg cells from the samehealthy volunteer.

In order to study the influence of a substance, e.g. HIV-1 gp120 whichcan bind at least to the epitope given in SEQ ID NO.:1 exclusively onthe suppressive function of CD4⁺CD25⁺ Treg cells, we developed aco-culture assay which contained CD8⁺ T cells as effectors to excludeany influence of this substance on the latter (CD8⁺ T cells don'texpress CD4). In this setting, activation of alloreactive CD8⁺ Teffector cells is only suppressed by activated CD4⁺CD25⁺ Treg cells suchas upon additional anti-CD3 mAb stimulation (positive control). Toevaluate the influence of e.g. HIV-1 gp120 on the function of CD4⁺CD25⁺Treg cells, isolated CD4⁺CD25⁺ Treg cells (2.1.4) are co-cultured withsyngenic, T cell-depleted and irradiated (50 Gy) PBMC 2.1.6) andallogeneic CD8⁺ T effector cells (2.1.3) in presence of varyingconcentrations (0.1-10 μg/ml) of different HIV-1 gp 120 preparations.Briefly, 1×10⁵ CD4⁺CD25⁺ Treg cells are incubated with 3×10⁵ syngenic Tcell-depleted PBMC in the presence or absence of varying amounts ofHIV-1 gp120. Stimulation with 0.5 μg/ml anti-CD3 monoclonal antibody(OKT-3, ebioscience, USA) serves as positive control. No additionalstimulation represents negative control. Either immediately or 24 h.later, 1×10⁵ allogeneic CD8⁺ T effector cells are added to the culturesand proliferation is determined after additional 72 h. by ³H-Tdrincorporation (37 kBq/well). Functional activation of CD4⁺CD25⁺ Tregcells via interaction with the epitope as set forth in SEQ ID NO:1result in suppressed proliferation of CD8⁺ T effector cells andtherewith identifies a substance as a Treg cell activator (Data see FIG.1).

To show in parallel whether a substance can interfere with HIV-1 gp120binding to CD4 epitope of a CD4⁺CD25⁺ Treg cell, CD4 is added indifferent concentrations (0.1-10 μg/ml) to isolated CD4⁺CD25⁻ Treg cells(2.1.4) cocultured with syngenic T cell-depleted PBMC (2.1.6) andallogeneic CD8⁺ T effector cells (2.1.3) in the presence of varyingamounts of the substance. Enhanced proliferation of CD8⁺ effector cellsresembles competitive binding to CD4 and blocked activation of a Tregcell via interaction with the epitope as set forth in SEQ ID NO:1. Allcultures are performed in serum free X-VIVO-15 (Cambrex, Verviers,Belgium).

Alternatively to irradiation-induced inactivation and block ofproliferation PBMC can be treated with Mitomycin C (Sigma, Germany).Briefly, 3×10⁷ PBMC are incubated in 3 ml MEM/10% FCS/180 μg Mitomycin Cfor 30 min at 37° C. Afterwards cells are washed 5× using MEM/10% FCS.Subsequently cells are subjected to the assay.

(2.2.3) Coculture suppression assay C: Stimulation of T cells inpresence of pre-activated CD4⁺CD25⁺ Treg cells and allogeneic PBMC

To evaluate the direct Treg cell activating potential of a compound inthe absence of antigen-presenting cells such as PBMC, isolated CD4⁺CD25⁺Treg cells (according to 2.1.4) are pre-cultured in X-VIVO-15 for 16-48h. alone, in presence of 0.5 μg/ml anti-CD3 monoclonal antibody (OKT-3)as positive control, or in presence of different concentrations of HIV-1gp120. Afterwards, cells are washed intensively and added to coculturesof syngenic, irradiated (50 Gy) PBMC and allogeneic CD4⁺ T helper cellsor CD8⁺ T effector cells. Proliferation is determined after additional72 h. by ³H-Tdr incorporation (37 kBq/well). Functional activation ofCD4⁺CD25⁺ Treg cells via interaction with the epitope as set forth inSEQ ID NO.: 1 result in suppressed proliferation of CD4⁺ T helper cellsor CD8⁺ T effector cells and therewith identifies a substance as a Tregcell activator.

A reduced incorporated radioactivity resembles suppressed proliferationand identifies CD8⁺ inhibition and therewith identifies a substance toactivate a Treg cell via interaction with the epitope as set forth inSEQ ID NO.: 1.

(2.3) Method for readout of suppressive activity of CD4⁺CD25⁺ Treg cells

(2.3.1) Analysis of proliferation

After 3-4 days of incubation of cells in assays provided under (2.2.1),(2.2.2) and (2.2.3) T cell proliferation is measured. For additional 16h., cells are pulsed with ³H-Tdr (37 kBq/well, MB Biomedicals), andincorporated radioactivity is measured by using a liquid scintillationcounter (Betaplate, Wallac/PerkinElmer). In the absence of a Treg cellactivating compound, resting Treg cells cannot inhibit the proliferationof CD4⁺ T helper cells or CD8⁺ T effector cells (negative control). Incontrast, in presence of an activating CD4-binding compound or anti-CD3monoclonal antibody (positive control) the proliferation of CD4⁺ Thelper cells or CD8⁺ T effector cells is suppressed by CD4⁺CD25⁺ Tregcells. A reduced incorporated radioactivity resembles suppressedproliferation and identifies CD4⁺ T helper cell or CD8⁺ T effector cellinhibition and therewith identifies that a CD4⁺CD25⁺ Treg cell has beenactivated.

For some experiments the proliferation of either CD4⁺CD25⁺ Treg cells,CD4⁺ T helper cells or CD8⁺ T effector cells is selectively followed byflow cytometry after labeling of these population with Vibrant CFDA SECell Tracer Kit (Invitrogen Life Technologies, San Diego, USA) accordingto the manufacturer's protocol. For CFDA labeling, T cells (1×10⁷cells/ml) are incubated in PBS with 1 μM CFDA Vibrant CFDA SE CellTracer Kit (Invitrogen Life Technologies, San Diego, USA) at 37° C. for30 min. Thereafter, cells are washed with X-VIVO-15 and incubated for 30min. at 37° C. in the dark. After an additional wash the cells arecounted and added to the cocultures. In addition, proliferation of cellsis measured by flow cytometry after 4-6 days of culture.

(2.3.2) Analysis of cytokine production

Activated Treg cells not only inhibit the proliferation of coculturedCD4⁺ T helper cells or CD8⁺ T effector cells, they suppress also thecytokine synthesis of these T cells. Therefore, detection of cytokinesproduced by cocultured T cells is an alternative method to analyze theTreg cell activating potential of a reagent. In this assay, isolatedCD4⁺CD25⁺ Treg cells (according to 2.1.4), syngenic CD3-depleted PBMC(according to 2.1.6) and allogeneic CD8⁺ T effector cells (according to2.1.3) are cocultured as described (2.2) before. After 7 days,alloreactive CD8⁺ T effector cells are polyclonal restimulated with 2.4μg/ml phytohemagglutinin (PHA, Sigma, Germany) and 1 ng/ml PMA for 5 h.in presence of monensin (BD GolgiStop™, BD Biosciences Pharmingen 1.3μM). Thereafter, cells are collected, washed with PBS, fixed andpermeabilized according to the manufacturer's instruction (perm/fixsolution, BD PharMingen, Germany) and stained with 0.5 μg/test of acytokine-specific monoclonal antibody (anti-IFN-γ, anti-IL-2,anti-TNF-α, all from BD PharMingen). Subsequently, production ofcytokines by CD4⁺ T helper cells or CD8⁺ T effector cells is analyzed byflow cytometry. In the absence of a Treg cell activating compound Tregcells cannot inhibit the cytokine production of CD4⁺ T helper cells orCD8⁺ T effector cells (negative control). In contrast, in presence of anactivating CD4-binding compound or anti-CD3 monoclonal antibodies(positive control) the cytokine production of CD4⁺ T helper cells orCD8⁺ T effector cells is suppressed by functional activated Treg cells.A reduced cytokine production identifies inhibition and therewithidentifies that a CD4⁺CD25⁺ Treg cell has been activated.

(2.3.3) Analysis of CD25 expression

Activated Treg cells inhibit the ability of CD8⁺ T effector cells andCD4⁺ T helper cells to express the α-chain of the IL-2 receptor, CD25.Therefore, the analysis of CD25 expression by flow cytometry is anadditional method to evaluate the Treg cell activating potential of areagent. In this assay, isolated CD4⁺CD25⁺ Treg cells (according to2.1.4) and syngenic CD3-depleted PBMC (according to 2.1.6) arecocultured with allogeneic CD8⁺ T effector cells or allogeneic CD4⁺ Thelper cells (according to 2.1.3, 2.1.2 and 2.2.1, 2.2.2 and 2.2.3).After 7 days, alloreactive CD8⁺ T effector cells or allogeneic CD4⁺ Thelper cells are stimulated with allogeneic PBMC from the same donor orDC as used in primary culture and expression of CD25 on alloreactiveCD8⁺ T effector or allogeneic CD4⁺ T helper cells cells are analyzed 24h. thereafter by flow cytometry. Activation of Treg cells by the testcompound results directly in an inhibited CD25 expression on there-stimulated CD8⁺ T effector cells or CD4⁺ T helper cells. In theabsence of a Treg cell activating compound, Treg cells cannot inhibitthe CD25 expression on re-stimulated CD8⁺ T effector cells or CD4⁺ Thelper cells (negative control). In contrast, in presence of anactivating reagent or an anti-CD3 monoclonal antibody (positive control)the CD25 expression of CD8⁺ T effector cells or CD4+ T helper cells issuppressed by Treg cells. A reduced CD25 expression identifiesinhibition and therewith identifies that a CD4⁺CD25⁺ Treg cell has beenactivated.

(2.3.4) Analysis of cyclic AMP production

Stimulation of Treg cells results in strong increase of cytosolic (i.e.intracellular) cAMP. Therefore, the analysis of cAMP in Treg cells is anadditional method to determine whether a substance which can interferewith HIV-1 gp120 binding to CD4 epitope of a CD4-positive cell canactivate a Treg cell. In this assay freshly isolated CD4⁺CD25⁺ Tregcells (according to 2.1.4) (1×10⁵-1×10⁶/well) are incubated withanti-CD3 monoclonal antibody (OKT-3; 0.5 μg/ml) or HIV-1 gp120 (0.1-10m/ml; Protein Science Corp., Meriden, Conn., USA) or left untreated for16 hours. To assess cytosolic cAMP-concentrations a cAMP-specific ELISA(Parameter™ Cyclic AMP Assay, Cat. No. KGE002; R&D Systems, Wiesbaden,Germany) is applied. Treg cells are washed three times in ice-cold PBSand subsequently lysed (1×10⁷/ml) using lysis buffer supplied by themanufacturer and applied to the ELISA according to the manufacturer'srecommendations.

Activation of Treg cells by the test compound results directly in anincrease of cytosolic cAMP. In the absence of a Treg cell activatingcompound no increase of cAMP in CD4⁺CD25⁺ Treg cells is detected(negative control). In contrast, in presence of an activatingCD4-binding compound or an anti-CD3 monoclonal antibody (positivecontrol) CD4⁺CD25⁺ Treg cell show a strong increase of cytosolic cAMP.An increase of cytosolic cAMP identifies that a CD4⁺CD25⁺ Treg cell hasbeen activated. Data see FIG. 2.

Alternatively, additional analysis of assay supernatant cAMPconcentration and use of phoshodiesterase inhibitors e.g. Roflumilast(1-50 μM) enhance the cAMP signal in the assay.

(3) In vivo assay for determining whether HIV-1 gp120 or a substancewhich can interfere with HIV-1 gp120-binding to a CD4⁺CD25⁺ Treg canactivate a CD4⁺CD25⁺ Treg in a disease-related model and therewith canbe useful in the treatment of an autoimmune disease (e.g. InflammatoryBowel Disease, Multiple Sclerosis, Rheumatoid Arthritis, Psoriasis,Diabetes Type I, Lupus Erythematosus, Phemphigus vulgaris,Thyreoiditis), other diseases with autoimmune aspects in theirpathogenesis such as vitiligo, atopic dermatitis, an allergy (e.g.Allergic rhinitis,), asthma (e.g. allergic asthma), GVHD(graft-versus-host disease), graft rejection.

(3.1) Method for induction of GVHD by transfer of human PBMC intoNOD-Scid mice and measurement of disease severity.

NOD-Scid mice, non-obese diabetic/severe combined immunodeficient mice(mutant allele: Prkdc^(Scid), strain: NOD.CB17-Prkdc^(Scid), (Shultz etal. 1995)) from the Central Laboratory Animal Facility of the Universityof Mainz (also purchasable from The Jackson Laboratory via Charles RiverLaboratories, Germany) are used as an animal model for transfer ofperipheral human immune cells and thus for the in vivo analysis ofimmuno-regulatory interactions (Hesselton et al. 1995). Human peripheralblood mononuclear cells (PBMC) transferred into NOD-Scid mice induce anaccelerating age-dependent graft-versus-host disease (GVHD). While thenumber of cells transferred determines the onset of this disease, theco-transfer of human Tregs can gradually delay or prevent the disease.This model system further allows the study of human Treg function. Toinduce a GVHD NOD-Scid mice at the age of three to six days after birthare intraperitoneally injected with 1×10⁷ to 3×10⁷ human PBMC (isolationof human PBMC according to 2.1.1). GVHD is induced by immunological(xenogenic) activation of human effector T cells in the PBMC fractioninjected into the mice. NOD-Scid mice, however, are not able to reactagainst the grafted human PBMC. The human PBMC-induced disease ischaracterized by lack of weight increase and weight loss, decreasedmobility, hunched posture, ruffled fur and organ inflammation in thetreated animals (Kizilisik and Al-Sebayel 1997). Transfer of human PBMCresults in growth arrest or weight loss within 30 to 40 days (dependingon the number of cells transferred) after transfer compared to untreatedmice. Lack of weight increase/weight loss is used as a parameter toscore GVHD severity. Control mice did not receive any PBMC. Data seeFIG. 3.

(3.2) Method for induction of GVHD by transfer of human PBMC intoNOD-Scid mice and prevention of disease by additional transfer of humanregulatory T cells and measurement of disease severity.

To prevent a GVHD disease in NOD-Scid mice at the age of three to sixdays after birth induced by intraperitoneally injection of 1×10⁷ to3×10⁷ human PBMC (isolation of human PBMC according to 2.1.1) additional2.5×10⁶ human regulatory T cells (isolation of human regulatory T cellsaccording to 2.1.4) are injected intraperitoneally (ratio Tregs to PBMC:1:4). Co-transfer of human regulatory T cells along with human PBMC(enhancement of Treg ratio) results in prevention of development of GVHDand weight loss similar development as untreated mice. Lack of weightincrease/weight loss is used as a parameter to score GVHD severity.Control mice did not receive any PBMC. Data see FIG. 3.

(3.3) Method for induction of GVHD by transfer of human PBMC intoNOD-Scid mice and prevention of disease by additional administration ofHIV-1 gp120 or a substance which can interfere with HIV-1 gp120-bindingand measurement of disease severity.

To prevent a GVHD disease induced in NOD-Scid mice at the age of threeto six days after birth induced by intraperitoneally injection of 1×10⁷to 3×10⁷ human PBMC (isolation of human PBMC according to 2.1.1) HIV-1gp120 or a substance which can interfere with HIV-1 gp120-binding isadministered in addition to the injection of human PBMC. Administrationof HIV-1 gp120 or a substance which can interfere with HIV-1gp120-binding induces an activation of human regulatory T cells in thePBMC fraction resulting in prevention of organ inflammation andprevention of growth arrest/weight loss similar to mice treated withhuman PBMC and additional human regulatory T cells or untreated mice.Lack of weight increase/weight loss is used as a parameter to score GVHDseverity. Control mice did not receive any PBMC. Data see FIG. 4.

(3.4) Method for induction of GVHD by transfer of human PBMC intoNOD-Scid mice and to demonstrate that prevention of disease byadministration of HIV-1 gp120 or a substance which can interfere withHIV-1 gp120-binding is mediated by human regulatory T cells in the PBMCfraction injected into NOD-Scid mice and measurement of diseaseseverity.

NOD-Scid mice at the age of three to six days after birth areintraperitoneally injected with 1×10⁷ human CD25-depleted PBMC(isolation of human CD25-depleted PBMC according to 2.1.7). GVHD isinduced by 1×10⁷ human CD25-depleted PBMC at the same magnitude as bytransfer of 1×10⁷ human non CD25-depleted PBMC into NOD-Scid mice. (Datasee FIG. 5 and FIG. 3 and FIG. 4) Mice receiving CD25-depleted PBMC andan additional administration of HIV gp120 are not protected fromdevelopment of GVHD and weight loss. Prevention of GVHD by HIV gp120 ora substance which can interfere with HIV-1 gp120-binding depends onregulatory T cells. Lack of weight increase/weight loss is used as aparameter to score GVHD severity. Data see FIG. 5.

DESCRIPTION OF FIGURES

FIG. 1: HIV-1 gp120 treatment activates human CD4⁺CD25⁺ Treg cells

In order to study the influence of HIV-1 gp120 exclusively on thefunction of CD4⁺CD25⁺ Treg cells, we developed a coculture assay whichcontained CD8⁺ T cells as effectors to exclude any influence of HIV-1gp120 on the latter. Therefore, isolated CD4⁺CD25⁺ Treg cells arecocultured with T cell-depleted syngenic PBMC and allogeneic CD8⁺ Teffector cells. In this setting, proliferation of alloreactive CD8⁺ Teffector cells is only suppressed by activated CD4⁺CD25⁺ Treg cells suchas upon additional anti-CD3 mAb stimulation or an activating HIV-1gp120-mediated signal. (All HIV-1 gp120 proteins: Protein Sciences Corp.Meriden, USA).

1×10⁵ isolated CD25⁺ Treg cells were incubated with 3×10⁵ irradiated (50Gy) syngenic T cell-depleted PBMC and 1×10⁵ allogeneic CD8⁺ T effectorcells in presence/absence of different HIV-1 gp120 preparations,addition of 0.5 μg/ml anti-CD3 (OKT-3) served as positive control.Proliferation was determined by ³H-Tdr-Incorporation 4 days later.

Grey bars represent proliferation of CD8⁺ T cells and inactivatedsyngenic CD3-depleted PBMC, white bars represent the proliferation ofCD4⁺CD25⁺ Tregs and the black bars represent the proliferation of CD8⁺ Tcells co-cultured with CD4⁺CD25⁺ Tregs cells and inactivated syngenicCD3-depleted PBMC. Due to inactivation PBMC don't contribute toproliferation of the samples.

As can be seen by the low white bars, Tregs don't show a significantproliferation under all conditions (without additional stimulus, oranti-CD3, or HIV-1 gp120 MN, or HIV-1 gp120 LAV, or HIV-1 gp120 CM).CD8⁺ T cells (grey bars) and CD8⁺ T cells co-cultured with Treg cells(black bars) show the same magnitude of proliferation under thecondition without additional stimulus representing the controls. Uponanti-CD3 stimulation CD8⁺ T cells (grey bars) show strong increasedproliferation but CD8⁺ T cells co-cultured with Treg cells (black bars)show a reduced proliferation indicating suppressed proliferation of CD8⁺T cells by anti-CD3 activated and therefore suppressive Tregs. Theeffect is anti-CD3 dose-dependent. Upon stimulation with HIV-1 gp120 MN,or HIV-1 gp120 LAV, or HIV-1 gp120 CM CD8⁺ T cells co-cultured with Tregcells (black bars) in contrast to CD8⁺ T cells without Tregs (grey bars)reduced proliferation indicating HIV-1 gp120 activated Tregs. Thisdemonstrates clearly that HIV-1 gp120 activates Tregs which subsequentlyexert suppressive activity on CD8⁺ T cells by reducing theirproliferation. The suppressive activity of Treg cells and therefore thereduced proliferation is HIV-1 gp120 dose-dependent.

FIG. 2: Cyclic AMP measurement in CD4+CD25+ Treg cells.

HIV-1 gp120 treatment augments cytosolic cAMP in human CD4⁺CD25⁺ Tregcells.

CD4⁺CD25⁺ regulatory T cells (CD25) are left untreated (Ø) or stimulatedwith an anti-CD3 monoclonal Antibody (OKT-3; 1 μg/ml) or HIV-1 gp120 (14ml). Upon 16 hours of stimulation the CD4⁺CD25⁺ regulatory T cells arelysed (1×10⁷/ml) and the cytosolic cAMP concentration of 1×10⁶ T cellsis assessed using a cAMP-specific ELISA.

CD4⁺CD25⁺ regulatory T cells (CD25) are left untreated (Ø) or stimulatedwith an anti-CD3 monoclonal Antibody (OKT-3; 1 μg/ml) or HIV-1 gp120 (1μg/ml). Upon 16 hours of stimulation the CD4⁺CD25⁺ regulatory T cellsare lysed (1×10⁷/ml) and the cytosolic cAMP concentration of 1×10⁶ Tcells is assessed using a cAMP-specific ELISA.

Bars represent the amount of cytosolic (intracellular) cyclic AMP (cAMP)in untreated Treg cells or Treg cells treated with different stimuli.The upper bar represent Tregs left untreated (Ø) and shows the basallevel of intracellular cAMP. Upon anti-CD3 stimulation (OKT-3) Tregs areactivated and show an increase of intracellular cAMP compared to theuntreated control (upper bar) as demonstrated with the second bar.Treatment of Tregs with HIV-1 gp120 also induces an increase ofintracellular cAMP as demonstrated with the lower bar. This demonstratedthat activation of Treg cells with different stimuli induces an increaseof cytosolic cAMP which can be used as readout for activation.

FIG. 3: Induction of GVHD by transfer of PBMC into NOD-Scid mice andprevention of GVHD by additional transfer of regulatory T cells (Tregs).

Three to six days old NOD-Scid mice are intraperitoneally injected with1×10⁷ human PBMC without (circles) or together with 2.5×10⁶ humanregulatory T cells (triangles). Control mice (rhombi) did not receiveany PBMC. Mice having received PBMC develop a fatal GVHD, do not growand die. Animals having additionally received regulatory T cells areprotected from development of GVHD and develop normally (3 mice pergroup). Lack of weight increase/weight loss is used as a parameter toscore GVHD severity. The diagram shows the relative body weight atdifferent time points after transfer.

FIG. 4: Induction of GVHD by transfer of PBMC into NOD-Scid mice andprevention of GVHD by additional injection of HIV gp120.

Three to six days old NOD-Scid mice are intraperitoneally injected with1×10⁷ human PBMC without (circles) or together with 5 μg HIV gp120(triangles). Control mice (rhombs) did not receive any PBMC. Mice havingreceived PBMC develop a fatal GVHD, do not grow and die. Animals havingadditionally received HIV gp120 are protected from development of GVHDand develop normally (3 mice per group). Lack of weight increase/weightloss is used as a parameter to score GVHD severity. The diagram showsthe relative body weight at different time points after transfer.

FIG. 5: Prevention of GVHD in NOD-Scid mice transferred with PBMC byadditional injection of HIV gp120 depends on the presence of regulatoryT cells.

Three to six days old NOD-Scid mice are intraperitoneally injected with1×10⁷ human CD25-depleted PBMC without (circles) or together with 5 μgHIV gp120 (triangles). Control mice (rhombi) do not receive any PBMC (3mice per group). Mice having received CD25-depleted PBMC develop a fatalGVHD, do not grow and die. Animals having received 1×10⁷ humanCD25-depleted PBMC and additionally HIV gp120 are not protected fromdevelopment of GVHD, also do not grow and die. Lack of weightincrease/weight loss is used as a parameter to score GVHD severity. Thediagram shows the relative body weight at different time points aftertransfer.

LITERATURE

-   Arthos J, Deen K C, Chaikin M A, Formwald J A, Sathe G, Sattentau Q    J, Clapham P R, Weiss R A, McDougal J S, Pietropaolo C, et al.-   Identification of the residues in human CD4 critical for the binding    of HIV.-   Cell. 1989 57:469-81-   Bluestone J A, Tang Q.-   Therapeutic vaccination using CD4+CD25+ antigen-specific regulatory    T cells.-   Proc Natl Acad Sci USA. 2004 101 Suppl 2:14622-6-   Carrière, D., Vendrell, J. P., Fontaine, C., Reynes, J., Atoui, N.,    and Pau, B.-   CD4 V1 domain masking on lymphocytes from HIV-1-infected patients.-   In: Leucocyte Typing V: White Cell Differentiation Antigens, Volume    1 pp 475-476, Oxford University Press 1995, Editor: S. F. Schlossman    et al.-   Castagna A, Biswas P, Beretta A, Lazzarin A.-   The appealing story of HIV entry inhibitors: from discovery of    biological mechanisms to drug development.-   Drugs. 2005 65:879-904-   Culp J S, Johansen H, Hellmig B, Beck J, Matthews T J, Delers A,    Rosenberg M. Regulated expression allows high level production and    secretion of HIV-1 gp120 envelope glycoprotein in Drosophila    Schneider cells.-   Biotechnology (N Y). 1991 9:173-7-   Diamond D C, Sleckman B P, Gregory T, Lasky L A, Greenstein J L,    Burakoff S J.-   Inhibition of CD4+ T cell function by the HIV envelope protein,    gp120.-   J. Immunol. 1988 141:3715-7-   Fontenot J D, Rudensky A Y.-   A well adapted regulatory contrivance: regulatory T cell development    and the forkhead family transcription factor Foxp3.-   Nat Immunol. 2005 6:331-7-   Franke R, Hirsch T, Overwin H, Eichler J.-   Synthetic Mimetics of the CD4 Binding Site of HIV-1 gp120 for the    Design of Immunogens.-   Angew Chem Int Ed Engl. 2007; January 9; [Epub ahead of print]-   Hesselton R M, Greiner D L, Mordes J P, Rajan T V, Sullivan J L,    Shultz L D.-   High levels of human peripheral blood mononuclear cell engraftment    and enhanced susceptibility to human immunodeficiency virus type I    infection in NOD/LtSz-scid/scid mice.-   J Infect Dis. 1995 172: 974-982.-   Hoffmann P, Eder R, Kunz-Schughart L A, Andreesen R, Edinger M.-   Large-scale in vitro expansion of polyclonal human CD4(+)CD25high    regulatory T cells.-   Blood. 2004 104:895-903-   Horwitz D A, Zheng S G, Gray J D, Wang J H, Ohtsuka K, Yamagiwa S.    Regulatory T cells generated ex vivo as an approach for the therapy    of autoimmune disease.-   Semin Immunol. 2004 16:135-43-   Hunig T, Dennehy K.-   CD28 superagonists: mode of action and therapeutic potential.-   Immunol Lett. 2005 100:21-8-   Jeffs S A, McKeating J, Lewis S, Craft H, Biram D. Stephens P E,    Brady R L. Antigenicity of truncated forms of the human    immunodeficiency virus type 1 envelope glycoprotein.-   J Gen Virol. 1996 July; 77 (Pt 7):1403-10-   Jameson B A, Rao P E, Kong L I, Hahn B H, Shaw G M, Hood L E, Kent    S B. Location and chemical synthesis of a binding site for HIV-1 on    the CD4 protein.-   Science. 1988 240:1335-9-   Kizilisik T A, al-Sebayel M.-   Diagnosis and classification of the severity of graft versus host    disease after experimental small-bowel transplantation in small    animal models.-   Transplant Proc. 1997 29:3030-3-   Klatzmann D, Champagne E, Chamaret S, Gruest J, Guetard D, Hercend    T, Gluckman J C, Montagnier L.-   T-lymphocyte T4 molecule behaves as the receptor for human    retrovirus LAV.-   Nature. 1984 312:767-8-   Lasky L A, Groopman J E, Fennie C W, Benz P M, Capon D J, Dowbenko D    J, Nakamura G R, Nunes W M, Renz M E, Berman P W.-   Neutralization of the AIDS retrovirus by antibodies to a recombinant    envelope glycoprotein.-   Science. 1986 233:209-12-   Leonard C K, Spellman M W, Riddle L, Harris R J, Thomas J N, Gregory    T J-   Assignment of intrachain disulfide bonds and characterization of    potential glycosylation sites of the type 1 recombinant human    immunodeficiency virus envelope glycoprotein (gp120) expressed in    Chinese hamster ovary cells.-   J Biol Chem. 1990 265:10373-82-   Lohman K L, Attanasio R, Buck D, Carrillo M A, Allan J S, Kennedy R    C.-   Characteristics of murine monoclonal anti-CD4. Epitope recognition,    idiotype expression, and variable region gene sequence.-   J. Immunol. 1992 149:3247-53-   Markovic I, Clouse K A.-   Recent advances in understanding the molecular mechanisms of HIV-1    entry and fusion: revisiting current targets and considering new    options for therapeutic intervention.-   Curr HIV Res. 2004 2: 223-34-   Mizukami T, Fuerst T R, Berger E A, Moss B.-   Binding region for human immunodeficiency virus (HIV) and epitopes    for HIV-blocking monoclonal antibodies of the CD4 molecule defined    by site-directed mutagenesis.-   Proc Natl Acad Sci USA. 1988; 85:9273-   Moreland L W, Haverty T P, Wacholtz M C, Knowles R W, Bucy R P, Heck    L W Jr, Koopman W J.-   Nondepleting humanized anti-CD4 monoclonal antibody in patients with    refractory rheumatoid arthritis.-   J. Rheumatol. 1998 25:221-8-   Muesing M A, Smith D H, Cabradilla C D, Benton C V, Lasky L A, Capon    D J.-   Nucleic acid structure and expression of the human    AIDS/lymphadenopathy retrovirus.-   Nature. 1985 313:450-8-   Repke H, Gabuzda D, Palu G, Emmrich F, Sodroski J.-   Effects of CD4 synthetic peptides on HIV type I envelope    glycoprotein function.-   J. Immunol. 1992 149:1809-16-   Robinson D S.-   Regulation: the art of control? Regulatory T cells and asthma and    allergy.-   Thorax. 2004 59:640-3-   Roncarolo, M. G, Bacchetta, R, Bordignon C, Narula, S, Levings, M K-   Type 1 T regulatory cells.-   Immunol Rev. 2001 182:68-79-   Sakaguchi, S.-   Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in    immunological tolerance to self and non-self.-   Nature. 2005 6:345-352-   Shevach E M.-   CD4+CD25+ suppressor T cells: more questions than answers.-   Nat Rev Immunol. 2002 2:389-400.-   Shultz L D, Schweitzer L, Christianson S W, Gott B, Schweitzer I B,    Tennent B, McKenna S, Mobraaten L, Rajan T V, Greiner D L, Leiter E    H.-   Multiple defects in innate and adaptive immunologic function in    NOD/LtSz-scid mice.-   J. Immunol. 1995 154: 180-191.-   Starcich B R, Hahn B H, Shaw G M, McNeely P D, Modrow S, Wolf H,    Parks E S, Parks W P, Josephs S F, Gallo R C, et al.-   Identification and characterization of conserved and variable    regions in the envelope gene of HTLV-III/LAV, the retrovirus of    AIDS.-   Cell. 1986 Jun. 6; 45(5):637-48-   Tang Q, Henriksen K J, Boden E K, Tooley A J, Ye J, Subudhi S K,    Zheng X X, Strom T B, Bluestone J A.-   Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+    regulatory T cells.-   J. Immunol. 2003 171:3348-52-   Thornton A M, Shevach E M.-   CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell    activation in vitro by inhibiting interleukin 2 production.-   J Exp Med. 1998 188:287-96-   Weiner, H. L.-   Induction and mechanism of action of transforming growth    factor-beta-secreting Th3 regulatory cells.-   Immunol Rev. 2001 182:207-14-   Yang Q E, Stephen A G, Adelsberger J W, Roberts P E, Zhu W, Currens    M J, Feng Y, Crise B J, Gorelick R J, Rein A R, Fisher R J,    Shoemaker R H, Sei S.-   Discovery of small-molecule human immunodeficiency virus type 1    entry inhibitors that target the 120-binding domain of CD4.-   J. Virol. 2005 79:6122-33-   Zheng S G, Wang J H, Gray J D, Soucier H, Horwitz D A.-   CD4+ and CD8+ regulatory T cells generated ex vivo with IL-2 and    TGF-beta suppress a stimulatory graft-versus-host disease with a    lupus-like syndrome.-   J. Immunol. 2004 172:5213-21

What is claimed is:
 1. A CD4⁺CD25⁺ Treg cell activator which activatesCD4⁺CD25⁺ Treg cell via interaction with the CD4⁺CD25⁺ Treg cell epitopeas set forth in SEQ ID NO:1, said CD4⁺CD25⁺ Treg cell activator being apeptide, a polypeptide, or an antibody or a binding fragment thereofwith the proviso that the antibody or antibody fragment is not OKT4A,OKT4D, OKTcdr4a, MAX.12H5 and not Leu3.
 2. The CD4⁺CD25⁺ Treg cellactivator according to claim 1 wherein said CD4⁺CD25⁺ Treg cellactivator is a peptide comprising the amino acid sequence as set forthin SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ IDNO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, or SEQ ID NO.:
 10. 3. A method oftreating an individual in need thereof comprising administering aCD4⁺CD25⁺ Treg cell activator which activates CD4⁺CD25⁺ Treg cell viainteraction with the CD4⁺CD25⁺ Treg cell epitope as set forth in SEQ IDNO:1, wherein said CD4⁺CD25⁺ Treg cell activator is a peptide, apolypeptide, or an antibody or a binding fragment thereof with theproviso that the antibody or antibody fragment is not OKT4A, OKT4D,OKTcdr4a, MAX.12H5 and not Leu3.
 4. The method of treating an individualin need thereof according to claim 3 wherein said Treg cell activator isa peptide comprising the amino acid sequence as set forth in SEQ ID NO.:3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ IDNO.: 8, SEQ ID NO.: 9, or SEQ ID NO.:
 10. 5. The method of treating anindividual in need thereof according to claim 3 wherein said CD4⁺CD25⁺Treg cell activator is HIV-1 gp120.
 6. The method of treating anindividual in need thereof according to claim 3 wherein said CD4⁺CD25⁺Treg cell activator is selected from a group consisting of: NSC 13778,peptide2 which presents three HIV-1 gp120 fragments bound togetherthrough comformationally flexible scaffolds, monoclonal antibody OKT4A,monoclonal antibody OKT4D, monoclonal antibody OKTcdr4a, monoclonalantibody MAX. 12H5, and monoclonal antibody Leu3.
 7. Use of a CD4⁺CD25⁺Treg cell activator which activates CD4⁺CD25⁺ Treg cell via interactionwith the CD4⁺CD25⁺ Treg cell epitope as set forth in SEQ ID NO:1 for thepreparation of a medicament for the treatment of a disease selected froma group consisting of a non-autoimmune inflammatory disease, anautoimmune inflammatory disease, an inflammatory disease due to organtransplantation; a bone marrow transplantation, and a disease due toexogenously administered self or exogenously administered non-autologousrecombinant polypeptide, wherein said CD4⁺CD25⁺ Treg cell activator isan antibody or an antibody fragment capable of binding to the peptide asset forth in SEQ ID NO.:1 or a peptide comprising the amino acidsequence as set forth in SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5,SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, or SEQ IDNO.:
 10. 8. The use according to according to claim 7 wherein saidnon-autoimmune inflammatory disease is selected from a group consistingof: asthma, allergic asthma, respiratory allergy, allergicrhinoconjunctivitis, allergic alveolitis, contact allergy, atopicdermatitis, neurodermatitis, food allergy, graft-versus-host disease,non-autoimmune inflammatory bowel disease, acute respiratory distresssyndrome, acute inflammatory pancreatitis, burns, wound healing, skinscarring disorders, sarcoidosis, Behcet's disease, Sweet's syndrome. 9.The use according to according to claim 7 wherein said an autoimmuneinflammatory disease is selected from a group consisting of: rheumatoidarthritis, rheumatic fever, systemic lupus erythematosus, ulcerativecolitis, Crohn's disease, autoimmune inflammatory bowel disease,diabetes type I, gastritis, autoimmune atrophic gastritis, autoimmunehepatitis, Hashimoto's thyroiditis, thyreoiditis, multiple sclerosis,myasthenia gravis, autoimmune haemolytic anemia, Addison's disease,scleroderma, Goodpasture's syndrome, Guillain-Barre syndrome, Graves'disease, glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid,vitiligo, idiopathic leukopenia, Sjogren's syndrome, Wegener'sgranulomatosis.
 10. The use according to according to claim 7 whereinsaid CD4⁺CD25⁺ Treg cell activator is HIV-1 gp120.
 11. The use accordingto according to claim 7 wherein said CD4⁺CD25⁺ Treg cell activator isselected from a group consisting of: NSC 13778, peptide2 which presentsthree HIV-1 gp120 fragments bound together through comformationallyflexible scaffolds, monoclonal antibody OKT4A, monoclonal antibodyOKT4D, monoclonal antibody OKTcdr4a, monoclonal antibody MAX. 12H5, andmonoclonal antibody Leu3.
 12. A pharmaceutical composition comprising atleast one CD4⁺CD25⁺ Treg cell activator which activates CD4⁺CD25⁺ Tregcell via interaction with the CD4⁺CD25⁺ Treg cell epitope as set forthin SEQ ID NO:1 wherein said CD4⁺CD25⁺ Treg cell activator is a peptide,a polypeptide, or an antibody or a binding fragment thereof with theproviso that the antibody or antibody fragment is not OKT4A, OKT4D,OKTcdr4a, MAX.12H5 and not Leu3.
 13. The pharmaceutical compositionaccording to claim 12 wherein said CD4⁺CD25⁺ Treg cell activator is apeptide comprising the amino acid sequence as set forth in SEQ ID NO.:3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ IDNO.: 8, SEQ ID NO.: 9, or SEQ ID NO.:
 10. 14. A method for reducing orpreventing an unwanted immune reaction due to a exogenously administeredself or exogenously administered non-autologous recombinant polypeptidecomprising administering to a being in need thereof a suitable amount ofa pharmaceutical composition comprising a CD4⁺CD25⁺ Treg cell activatorwhich is an antibody or an antibody fragment capable of binding to thepeptide as set forth in SEQ ID NO.:1 or is a peptide comprising theamino acid sequence as set forth in SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ IDNO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, orSEQ ID NO.:
 10. 15. The method according to claim 14 wherein saidCD4⁺CD25⁺ Treg cell activator is HIV-1 gp120.
 16. The method accordingto claim 14 wherein said Treg cell activator is selected from a groupconsisting of: NSC 13778, peptide 2 which presents three HIV-1 gp120fragments bound together through comformationally flexible scaffolds,monoclonal antibody OKT4A, monoclonal antibody OKT4D, monoclonalantibody OKTcdr4a, monoclonal antibody MAX. 12H5, and monoclonalantibody Leu3.